Immunomodulatory compositions

ABSTRACT

Isolated immunomodulatory (e.g. immunostimulatory) polyhydroxlated pyrrolizidine compounds having the formula  
                 
are disclosed. In these compounds R is selected from hydrogen, straight or branched, unsubstituted or substituted, saturated or unsaturated acyl, alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups. The compounds are useful in therapy and prophylaxis, including increasing the Th1:Th2 response ratio, hemorestoration, alleviation of immunosuppression, cytokine stimulation, treatment of proliferative disorders (e.g. cancer), vaccination, stimulation of the innate immune response and boosting of the activity of endogenous NK cells.

FIELD OF THE INVENTION

The present invention relates to immunomodulatory polyhydroxylatedpyrrolizidine compounds and to their use in medicine. In particular, theinvention relates to the use of casuarine and certain casuarineanalogues as immunomodulatory (immunostimulatory or immunosuppressive)drugs.

BACKGROUND TO THE INVENTION

Immunity

When the immune system is challenged by a foreign antigen it responds bylaunching a protective response. This response is characterized by thecoordinated interaction of both the innate and acquired immune systems.These systems, once thought to be separate and independent, are nowrecognized as two interdependent parts that when integrated fulfill twomutually exclusive requirements: speed (contributed by the innatesystem) and specificity (contributed by the adaptive system).

The innate immune system serves as the first line of defence againstinvading pathogens, holding the pathogen in check while the adaptiveresponses are matured. It is triggered within minutes of infection in anantigen-independent fashion, responding to broadly conserved patterns inthe pathogens (though it is not non-specific, and can distinguishbetween self and pathogens). Crucially, it also generates theinflammatory and co-stimulatory milieu (sometimes referred to as thedanger signal) that potentiates the adaptive immune system and steers(or polarizes it) towards the cellular or humoral responses mostappropriate for combating the infectious agent (discussed in more detailbelow).

The adaptive response becomes effective over days or weeks, butultimately provides the fine antigenic specificity required for completeelimination of the pathogen and the generation of immunologic memory. Itis mediated principally by T and B cells that have undergone germlinegene rearrangement and are characterized by an exquisite specificity andlong-lasting memory. However, it also involves the recruitment ofelements of the innate immune system, including professional phagocytes(macrophages, neutrophils etc.) and granulocytes (basophils, eosinophilsetc.) that engulf bacteria and even relatively large protozoalparasites. Once an adaptive immune response has matured, subsequentexposure to the pathogen results in its rapid elimination (usuallybefore symptoms of infection become manifest) because highly specificmemory cells have been generated that are rapidly activated uponsubsequent exposure to their cognate antigen.

Interdependence of Innate and Adaptive Responses

It is now thought that the earliest events following pathogen invasionare effected by cellular components of the innate immune system. Theresponse is initiated when resident tissue macrophages and dendriticcells (DCs) encounter pathogen and become activated by signals generatedby interaction between pattern-recognition receptors (PRRs) and thepathogen-associated molecular patterns (PAMPs) shared by large groups ofmicroorganisms. The activated macrophages and DCs are stimulated torelease various cytokines (including the chemokines IL-8, MIP-1α andMIP-1β), which constitute the danger signal and triggers an influx ofNatural Killer (NK) cells, macrophages, immature dendritic cells intothe tissues.

Loaded with antigen, the activated DCs then migrate to lymph nodes. Oncethere, they activate immune cells of the adaptive response (principallynaïve B- and T-cells) by acting as antigen-presenting cells (APCs). Theactivated cells then migrate to the sites of infection (guided by the“danger signal”) and once there further amplify the response byrecruiting cells of the innate immune system (including eosinophils,basophils, monocytes, NK cells and granulocytes). This cellulartrafficking is orchestrated by a large array of cytokines (particularlythose of the chemokine subgroup) and involves immune cells of manydifferent types and tissue sources (for a review, see Luster (2002),Current Opinion in Immunology 14: 129-135).

Polarization of the Adaptive Immune Response

The adaptive immune response is principally effected via two independentlimbs: cell-mediated (type 1) immunity and antibody-mediated or humoral(type 2) immunity.

Type 1 immunity involves the activation of T-lymphocytes that either actupon infected cells bearing foreign antigens or stimulate other cells toact upon infected cells. This branch of the immune system thereforeeffectively contains and kills cells that are cancerous or infected withpathogens (particularly viruses). Type 2 immunity involves thegeneration of antibodies to foreign antigens by B-lymphocytes. Thisantibody-mediated branch of the immune system attacks and effectivelyneutralizes extracellular foreign antigens.

Both limbs of the immune system are important in fighting disease andthere is an increasing realization that the type of immune response isjust as important as its intensity or its duration. Moreover, since thetype 1 and type 2 responses are not necessarily mutually exclusive (inmany circumstances an effective immune response requires that both occurin parallel), the balance of the type 1/type 2 response (also referredto as the Th1:Th2 response ratio/balance by reference to the distinctcytokine and effector cell subsets involved in the regulation of eachresponse—see below) may also play a role in determining theeffectiveness (and repercussions) of the immune defence.

In many circumstances the immune response is skewed heavily towards atype 1 or type 2 response soon after exposure to antigen. The mechanismof this type 1/type 2 skewing or polarization is not yet fullyunderstood, but is known to involve a complex system of cell-mediatedchemical messengers (cytokines, and particularly chemokines) in whichthe type 1/type 2 polarization (or balance) is determined, at least inpart, by the nature of the initial PRR-PAMP interaction when the DCs andmacrophages of the innate immune system are first stimulated andsubsequently by the cytokine milieu in which antigen priming of naïvehelper T cells occurs.

Two cytokines in particular appear to have early roles in determiningthe path of the immune response. Interleukin-12 (IL-12), secreted bymacrophages, drives the type 1 response by stimulating thedifferentiation of Th1 cells, the helper cells that oversee the type 1response. Another macrophage cytokine, interleukin-10 (IL-10) inhibitsthis response, instead driving a type 2 response.

The type 1 and type 2 responses can be distinguished inter alia on thebasis of certain phenotypic changes attendant on priming and subsequentpolarization of naïve helper T cells. These phenotypic changes arecharacterized, at least in part, by the nature of the cytokines secretedby the polarized helper T cells.

Th1 cells produce so-called Th1 cytokines, which include one or more ofTNF, IL-1, IL-7, IFN-gamma, IL-12 and/or IL-18. The Th1 cytokines areinvolved in macrophage activation and Th1 cells orchestrate Type 1responses. In contrast, Th2 cells produce so-called Th2 cytokines, whichinclude one or more of IL-4, IL-5, IL-10 and IL-13. The Th2 cytokinespromote the production of various antibodies and can suppress the type 1response.

The involvement of Th1 and Th2 cells and cytokines in type 1:type 2immune response polarization has given rise to the terms Th1 responseand Th2 response being used to define the type 1 and type 2 immuneresponses, respectively. Thus, these terms are used interchangeablyherein.

There is an increasing realization that the type of immune response isjust as important in therapy and prophylaxis as its intensity or itsduration. For example, an excess Th1 response can result in autoimmunedisease, inappropriate inflammatory responses and transplant rejection.An excess Th2 response can lead to allergies and asthma. Moreover, aperturbation in the Th1:Th2 ratio is symptomatic of many immunologicaldiseases and disorders, and the development of methods for altering theTh1:Th2 ratio is now a priority.

Alkaloids

The term alkaloid is used herein sensu stricto to define any basic,organic, nitrogenous compound which occurs naturally in an organism. Theterm alkaloid is also used herein sensu lato to define a broadergrouping of compounds which include not only the naturally occurringalkaloids, but also their synthetic and semi-synthetic analogues andderivatives.

Most known alkaloids are phytochemicals, present as secondarymetabolites in plant tissues (where they may play a role in defence),but some occur as secondary metabolites in the tissues of animals,microorganisms and fungi. There is growing evidence that the standardtechniques for screening microbial cultures are inappropriate fordetecting many classes of alkaloids (particularly highly polaralkaloids, see below) and that microbes (including bacteria and fungi,particularly the filamentous representatives) will prove to be animportant source of alkaloids as screening techniques become moresophisticated.

Structurally, alkaloids exhibit great diversity. Many alkaloids aresmall molecules, with molecular weights below 250 Daltons. The skeletonsmay be derived from amino acids, though some are derived from othergroups (such as steroids). Others can be considered as sugar analogues.It is becoming apparent (see Watson et al. (2001) Phytochemistry 56:265-295) that the water soluble fractions of medicinal plants andmicrobial cultures contain many interesting novel polar alkaloids,including many carbohydrate analogues. Such analogues include a rapidlygrowing number of polyhydroxylated alkaloids.

Most alkaloids are classified structurally on the basis of theconfiguration of the N-heterocycle. Examples of some important alkaloidsand their structures are set out in Kutchan (1995) The Plant Cell7:1059-1070.

Watson et al. (2001) Phytochemistry 56: 265-295 have classified acomprehensive range of polyhydroxylated alkaloids inter alia aspiperidine, pyrroline, pyrrolidine, pyrrolizidine, indolizidine andnortropanes alkaloids (see FIGS. 1-7 of Watson et al. (2001), thedisclosure of which is incorporated herein by reference).

Watson et al. (2001), ibidem also show that a functional classificationof at least some alkaloids is possible on the basis of their glycosidaseinhibitory profile: many polyhydroxylated alkaloids are potent andhighly selective glycosidase inhibitors. These alkaloids can mimic thenumber, position and configuration of hydroxyl groups present inpyranosyl or furanosyl moieties and so bind to the active site of acognate glycosidase, thereby inhibiting it. This area is reviewed inLegler (1990) Adv. Carbohydr. Chem. Biochem. 48: 319-384 and in Asano etal., (1995) J. Med. Chem. 38: 2349-2356.

It has long been recognized that many alkaloids are pharmacologicallyactive, and humans have been using alkaloids (typically in the form ofplant extracts) as poisons, narcotics, stimulants and medicines forthousands of years. The therapeutic applications of polyhydroxylatedalkaloids have been comprehensively reviewed in Watson et al. (2001),ibidem: applications include cancer therapy, immune stimulation, thetreatment of diabetes, the treatment of infections (especially viralinfections), therapy of glycosphingolipid lysosomal storage diseases andthe treatment of autoimmune disorders (such as arthritis and sclerosis).

Both natural and synthetic mono- and bi-cyclic nitrogen analogues ofcarbohydrates are known to have potential as chemotherapeutic agents.Alexine (1) and australine (2) were the first pyrrolizidine alkaloids tobe isolated with a carbon substituent at C-3, rather than the morecommon C-1 substituents characteristic of the necine family ofpyrrolizidines.

The alexines occur in all species of the genus Alexa and also in therelated species Castanospermum australe. Stereoisomers of alexine,including 1,7a-diepialexine (3), have also been isolated (Nash et al.(1990) Phytochemistry (29) 111) and synthesised (Choi et al. (1991)Tetrahedron Letters (32) 5517 and Denmark and Cottell (2001) J. Org.Chem. (66) 4276-4284).

Because of the reported weak in vitro antiviral properties of one7,7a-diepialexine (subsequently defined as 1,7a-diepialexine), there hasbeen some interest in the isolation of the natural products and thesynthesis of analogues.

As an indolizidine alkaloid (and so structurally distinct from thepyrrolizidine alexines), swainsonine (4) is a potent and specificinhibitor of α-mannosidase and is reported to have potential as anantimetastic, tumour anti-proliferative and immunoregulatory agent (seee.g. U.S. Pat. No. 5,650,413, WO00/37465, WO93/09117).

The effect of variation in the size of the six-membered ring ofswainsonine on its glycosidase inhibitory activity has been studied:pyrrolizidine derivatives (so-called “ring contracted swainsonines”)have been synthesised. However, these synthetic derivatives(1S,2R,7R,7aR)1,2,7-trihydroxypyrrolizidine (5) and the 7S-epimer (6))were shown to have much weaker inhibitory activity relative toswainsonine itself (see U.S. Pat. No. 5,075,457).

Another compound, 1α,2α,6α,7α,7αβ-1,2,6,7-tetrahydroxypyrrolizidine (7)is an analogue of 1,8-diepiswainsonine and described as a “useful”inhibitor of glycosidase enzymes in EP0417059.

Casuarine,(1R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)-1,2,6,7-tetrahydroxypyrrolizidine(8) is a highly oxygenated bicyclic pyrrolizidine alkaloid that can beregarded as a more highly oxygenated analogue of the 1,7a-diepialexine(shown in 3) or as a C(3) hydroxymethyl-substituted analogue of the1α,2α,6α,7α,7αβ-1,2,6,7-tetrahydroxypyrrolizidine (shown in 7).

Casuarine can be isolated from several botanical sources, including thebark of Casuarina equisetifolia (Casuarinaceae), the leaves and bark ofEugenia janibolana (Myrtaceae) and Syzygium guineense (Myrtaceae) (seee.g. Nash et al. (1994) Tetrahedron Letters (35) 7849-7852). Epimers ofcasuarine, and probably casuarine itself, can be synthesised by sodiumhydrogen telluride-induced cyclisation of azidodimesylates (Bell et al.(1997) Tetrahedron Letters (38) 5869-5872).

Casuarina equisetifolia wood, bark and leaves have been claimed to beuseful against diarrhoea, dysentery and colic (Chopra et al. (1956)Glossary of Indian Medicinal Plants, Council of Scientific andIndustrial Research (India), New Delhi, p. 55) and a sample of bark hasrecently been prescribed in Western Samoa for the treatment of breastcancer. An African plant containing casuarine (identified as Syzygiumguineense) has been reported to be beneficial in the treatment of AIDSpatients (see Wormald et al. (1996) Carbohydrate Letters (2) 169-174).

The casuarine-6-α-glucoside (casuarine-6-α-D-glucopyranose, 9) has alsobeen isolated from the bark and leaves of Eugenia jambolana (Wormald etal. (1996) Carbohydrate Letters (2) 169-174).

Eugenia jambolana is a well known tree in India for the therapeuticvalue of its seeds, leaves and fruit against diabetes and bacterialinfections. Its fruit have been shown to reduce blood sugar levels inhumans and aqueous extracts of the bark are claimed to affectglycogenolysis and glycogen storage in animals (Wormald et al. (1996)Carbohydrate Letters (2) 169-174).

Dendritic Cells and their Immunotherapeutic Uses

(a) Introduction

Dendritic cells (DCs) are a heterogeneous cell population withdistinctive morphology and a widespread tissue distribution (seeSteinman (1991) Ann. Rev. Immunol. 9: 271-296). They play an importantrole in antigen presentation, capturing and processing antigens intopeptides and then presenting them (together with components of the MHC)to T cells. T cell activation may then be mediated by the expression ofimportant cell surface molecules, such as high levels of MHC class I andII molecules, adhesion molecules, and costimulatory molecules.

Dendritic cells therefore act as highly specialized antigen-presentingcells (APCs): serving as “nature's adjuvants”, they potentiate adaptiveT-cell dependent immunity as well as triggering the natural killer (NKand NKT) cells of the innate immune system. Dendritic cells thereforeplay a fundamental and important regulatory role in the magnitude,quality, and memory of the immune response. As a result, there is now agrowing interest in the use of dendritic cells in variousimmunomodulatory interventions, which are described in more detailbelow.

Dendritic cells can be classified into different subsets inter alia onthe basis of their state of maturation (mature or immature) and theircellular developmental origin (ontogeny). Each of these subsets appearto play distinct roles in vivo, as described below.

(b) Dendritic Cell Maturation

Immature (or resting) DCs are located in non-lymphoid tissue, such asthe skin and mucosae, are highly phagocytic and readily internalizesoluble and particulate antigens. It is only when such antigen-loadedimmature DCs are also subject to inflammatory stimuli (referred to asmaturation stimuli) that they undergo a maturation process thattransforms them from phagocytic and migratory cells into non-phagocytic,highly efficient stimulators of naïve T cells.

Immature DCs are characterized by high intracellular MHC II in the formof MIICs, the expression of CD1a, active endocytosis for certainparticulates and proteins, presence of FcgR and active phagocytosis,deficient T cell sensitization in vitro, low/absent adhesive andcostimulatory molecules (CD40/54/58/80/86), low/absent CD25, CD83, p55,DEC-205, 2A1antigen, responsiveness to GM-CSF, but not M-CSF and G-CSFand a sensitivity to IL-10, which inhibits maturation.

Upon maturation, mature DCs, loaded with antigen and capable of primingT cells, migrate from the non-lymphoid tissues to the lymph nodes orspleen, where they process the antigen load and present it to theresident naïve CD4⁺ T cells and CD8⁺ cytotoxic T cells. This latterinteraction generates CTLs, the cellular arm of the adaptive immuneresponse, and these cells eliminate virally infected cells and tumourcells. The naïve CD4⁺ T cells differentiate into memory helper T cells,which support the differentiation and expansion of CD8⁺ CTLs and Bcells. Thus, helper T cells exert anti-tumour activity indirectlythrough the activation of important effector cells such as macrophagesand CTLs.

Having activated the T cells in this way, the mature DCs undergoapoptosis within 9-10 days.

Mature DC cells are characterized morphologically by motility and thepresence of numerous processes (veils or dendrites). They are competentfor antigen capture and presentation (exhibiting high MHC class I and IIexpression) and express a wide range of molecules involved in T cellbinding and costimulation, (e.g. CD40, CD54/ICAM-1, CD58/LFA-3,CD80/B7-1 and CD86/B7-2) as well as various cytokines (including IL-12).They are phenotypically stable: there is no reversion/conversion tomacrophages or lymphocytes.

Thus, mature DCs play an important role in T cell activation andcell-mediated immunity. In contrast, immature DCs are involved inregulating and maintaining immunological tolerance (inducingantigen-specific T cell energy).

(c) Dendritic Cell Ontogenic Subsets

Dendritic cells are not represented by a single cell type, but rathercomprise a heterogeneous collection of different classes of cells, eachwith a distinct ontogeny. At least three different developmentalpathways have been described, each emerging from unique progenitors anddriven by particular cytokine combinations to DC subsets with distinctand specialized functions.

At present it is thought that the earliest DC progenitors/precursorscommon to all DCs originate in the bone marrow. These primitiveprogenitors are CD34⁺, and they are released from the bone marrow tocirculate through both the blood and lymphoid organs.

Once released from the bone marrow, the primitive CD34⁺ DC progenitorsare subject to various stimulatory signals. These signals can direct theprogenitors along one of at least three different pathways, eachdiffering with respect to intermediate stages, cytokine requirements,surface marker expression and biological function.

-   -   Lymphoid DCs are a distinct subset of DCs that are closely        linked to the lymphocyte lineage. This lineage is characterized        by the lack of the surface antigens CD11b, CD13, CD14 and CD33.        Lymphoid DCs share ancestry with T and natural killer (NK)        cells, the progenitors for all being located in the thymus and        in the T cell areas of secondary lymphoid tissues. The        differentiation of lymphoid DCs is driven by Interleukins 2, 3        and 15 (IL-3, IL-2 and IL-1), but not by granulocyte macrophage        colony-stimulating factor (GM-CSF). Functionally, lymphoid        promote negative selection in the thymus (possibly by inducing        fas-mediated apoptosis) and are costimulatory for CD4⁺ and CD8⁺        T cells. More recently, lymphoid-like DCs derived from human        progenitors have also been shown to preferentially activate the        Th2 response. Because of their capacity to induce apoptosis and        their role in eliminating potentially self-reactive T cells, it        has been suggested that lymphoid DCs primarily mediate        regulatory rather than stimulatory immune effector functions.    -   Myeloid DCs are distinguished by a development stage in which        there is expression of certain features associated with        phagocytes. There appear to be at least two structurally and        functionally distinct subsets. The first is defined        antigenically as CD14⁻, CD34⁺, CD68⁻ and CD1a⁺ and sometimes        referred to as DCs of the Langerhans cell type. This subset        appears to prime T cells to preferentially activate Th1        responses and IL-12 appears implicated in this process. The        subset may also activate naïve B cells to secrete IgM and may        therefore be predominantly associated with an inflammatory Th1        response. A second myeloid DC subset, sometimes referred to as        interstitial DCs, is defined antigenically as CD14⁺, CD68⁺ and        CD1a⁻ and related to monocytes (as a result they are also        referred to as monocyte-derived DCs or Mo-DCs).        (d) Dendritic Cell Vaccines

In one dendritic cell-based treatment paradigm (reviewed in Schuler etal. (2003) Current Opinion in Immunol 15: 138-147), DC cells are takenfrom a patient (for example by apheresis) and then pulsed (primed orspiked) with a particular antigen or antigens (for example, tumourantigen(s)). They are then re-administered as an autologous cellularvaccine to potentiate an appropriate immune response.

In this treatment paradigm, the responding T cells include helper cells,especially Th1 CD4⁺ cells (which produce IFN-γ) and killer cells(especially CD8⁺ cytolytic T lymphocytes). The DCs may also mediateresponses by other classes of lymphocytes (B, NK, and NKT cells). Theymay also elicit T cell memory, a critical goal of vaccination.

At present, little is known about the identity of the DC subset(s)required for optimum effectiveness of DC vaccines, beyond therecognition that maturation is required and immature DCs are to beavoided (Dhodapkar and Steinman (2002) Blood 100: 174-177).

Hsu et al. (1996) Nat Med 2: 52-58 used rare DCs isolated ex vivo fromblood. These DCs were highly heterogeneous with respect to theirontogenic subsets but matured spontaneously during the isolationprocedure. However, the yields were very low.

The yield problem has been addressed by the development of techniquesfor expanding the DCs ex vivo, for example with Flt3 ligand (Fong et al.(2001) PNAS 98: 8809-8814), but this is of limited effectiveness.

However, most studies have used Mo-DCs. These cells are obtained byexposing monocytes to GM-CSF and IL-4 (or IL-13) to produce immatureMo-DCs, which are then matured by incubation in a maturation medium.Such media comprise one or more maturation stimulation factor(s), andtypically comprise Toll-like receptor (TLR) ligands (e.g. microbialproducts such as lipopolysaccharide and/or monophosphoryl lipid),inflammatory cytokines (such as TNF-α), CD40L, monocyte conditionedmedium (MCM) or MCM mimic (which contains IL-1β, TNF-α, IL-6 and PGE₂).

Although little is known at present about the influence of maturationmedium on DC vaccine performance, MCM or MCM mimic currently represent astandard: Mo-DCs matured using these media are homogenous, have a highviability, migrate well to chemotactic stimuli and induce CTLs both invitro and in vivo.

Techniques have been developed for generating large numbers of Mo-DCs(300 to 500 million mature DCs per apheresis) from adherent monocyteswithin semi-closed, multilayered communicating culture vessels offeringa surface area large enough to cultivate one leuk-apheresis product.These so-called cell factories can be used to produce cryopreservedaliquots of antigen preloaded DCs which are highly viable on thawing,and optimised maturation and freezing procedures have been described(Berger et al. (2002) J. Immunol. Methods 268: 131-140; Tuyaerts et al.(2002) J. Immunol. Methods 264: 135-151).

Dendritic cells for vaccination have also been prepared fromCD34⁺-derived DCs comprising a mixture of interstitial and DCs of theLangerhans cell type. Some workers believe that the latter DC subset aremore potent than Mo-DCs when used as DC vaccines.

With regard to antigen selection, various approaches have been used.Both defined and undefined antigens can be employed. The antigens can bexenoantigens or autoantigens. One or more defined neoantigen(s) may beselected: in the case of cancer treatment, the neoantigen(s) maycomprise a tumour-associated antigen. However, most popular are 9-11amino acid peptides containing defined antigens (either naturalsequences or analogues designed for enhanced MHC binding): such antigenscan be manufactured to good manufacturing practice (GMP) standard andare easily standardized.

Other approaches have employed antigens as immune complexes, which aredelivered to Fc-receptor-bearing DCs and which results in the formationof both MHC class I and MHC class II peptide sequences. This offers thepotential for inducing both CTLs and Th cells (Berlyn et al. (2001) ClinImmunol 101: 276-283).

Methods have also been developed for exploring the whole antigenicrepertoire of any given tumour (or other target cell, such as avirally-infected cell). For example, DC-tumour cell hybrids have beensuccessfully used to treat renal cell carcinoma (Kugler et al. (2000) 6:332-336), but the hybrids are difficult to standardize and short-lived.Necrotic or apoptotic tumour cells have been used, as have variouscellular lysates.

It appears that the selection of patient-specific antigens may beimportant in the treatment of at least some cancers, and antigensderived from fresh tumour cells rather than tumour cell lines or definedantigens may prove important (Dhodapkar et al. (2002) PNAS 99:13009-13013).

As regards delivery of the selected antigen(s) to the DCs, varioustechniques are available. Since the number and quality of MHC-peptidecomplexes directly influences the immunogenicity of the DC, the antigenloading technique may prove critical to DC vaccine performance (van derBurg et al. (1996) J Immunol 156: 3308-3314). It seems that prolongedpresentation of MHC-peptide complexes by the DCs enhances immunogenicityand so loading techniques which promote prolonged presentation may beimportant. This has been achieved by loading the DCs internally throughthe use of peptides linked to cell-penetrating moieties (Wang and Wang(2002) Nat Biotechnol 20: 149-154).

Antigens can also be loaded by transfecting the DCs with encodingnucleic acid (e.g. by electroporation) such that the antigens areexpressed by the DC, processed and presented at the cell surface. Thisapproach avoids the need for expensive GMP proteins and antibodies. RNAis preferred for this purpose, since it produces only transientexpression (albeit sufficient for antigen processing) and avoids thepotential problems associated with the integration of DNA and attendantlong-term expression/mutagenesis. Such transfection techniques alsopermit exploration of the whole antigenic repertoire of a target cell byuse of total or PCR-amplified tumour RNA.

There is some evidence that helper proteins (for example, keyhole limpethemocyanin (KLH) and tetanus toxoid (TT)) can provide unspecific helpfor CTL induction (Lanzavecchia (1998) Nature 393: 413-414) and it mayprove advantageous to pulse DC with such helper proteins prior tovaccination.

With regard to posology, the dose, frequency and route of DC vaccineadministration have not yet been optimised in clinical trials. Clearly,the absolute number of cells administered will depend on the route ofadministration and effectiveness of migration after infusion. In thisrespect there are indications that intradermal or subcutaneousadministration may be preferred for the development of Th1 responses,although direct intranodal delivery has been employed to circumvent theneed for migration from the skin to the nodes (Nestle et al. (1998) NatMed 4: 328-332).

Quite distinct from the antigen-pulsed DC vaccine paradigm describedabove is an approach in which dendritic cells secreting variouschemokines are injected directly into tumours where they have been shownto prime T cells extranodally (Kirk et al. (2001) Cancer Res 61:8794-8802). Thus, in another treatment paradigm, DCs are targeted to atumour and activated to elicit immune responses in situ without the needfor ex vivo antigen loading.

In situ DC vaccination constitutes yet another distinct (but related)approach (Hawiger et al. (2001) J Exp Med 194: 769-779. In thistherapeutic paradigm, antigen is targeted to DCs in vivo which areexpanded and induced to mature in situ. This approach depends onefficient targeting of antigen to endogenous DCs (for example, usingexosomes—see Thery et al. (2002) Nat Rev Immunol 2: 569-579) and thedevelopment of maturation stimulants that can effectively triggermaturation (preferably of defined DC subset(s)) in vivo.

(e) Use of Dendritic Cells in Adoptive CTL Immunotherapy

Cytotoxic T lymphocytes (CTLs) can be administered to a patient in orderto confer or supplement an immune response to a particular disease orinfection (typically cancer). For example, tumour specific T cells canbe extracted from a patient (e.g. by leukapheresis), selectivelyexpanded (for example by tetramer-guided cloning—see Dunbar et al.(1999) J Immunol 162: 6959-6962) and then re-administered as anautologous cellular vaccine.

The clinical effectiveness, applicability and tractability of this typeof passive immunotherapy can be greatly increased by using dendriticcells to prime the T cells in vitro prior to administration.

(f) Dendritic Cell-Based Approaches to the Treatment of AutoimmuneDisorders

Dendritic cells are also involved in regulating and maintainingimmunological tolerance: in the absence of maturation, the cells induceantigen-specific silencing or tolerance.

Thus, in another dendritic cell-based treatment paradigm, immature DCsare administered as part of an immunomodulatory intervention designed tocombat autoimmune disorders. In such applications, the suppressivepotential of the DCs has been enhanced by in vitro transfection withgenes encoding cytokines.

(g) The Role of IL-2 in Dendritic Cell Function

Granucci et al. (2002) Trends in Immunol. 23: 169-171 have reportedtransient upregulation of mRNA transcripts for IL-2 in dendritic cellsfollowing microbial stimulus. In WO03012078 Granucci describes theimportant role played by DC-derived IL-2 in mediating not only T cellactivation but also that of NK cells and goes on to suggest thatDC-derived IL-2 is a key factor regulating and linking innate andadaptive immunity.

Moreover, systemic administration of IL-2 has recently been shown toenhance the therapeutic efficacy of a DC vaccine (Shimizu et al. (1999)PNAS 96: 2268-2273), while the presence of IL-2 was shown to beessential for specific peptide-mediated immunity mediated by dendriticcells in at least some DC vaccination regimes (Eggert et al. (2002) EurJ Immunol 32: 122-127). In their recent review, Schuler et al. (ibidem)conclude that “ . . . it might be worthwhile to explore the combinationof DC vaccination with IL-2 administration, as the T-cell responsesinduced by DC vaccination appear enhanced and therapeutically moreeffective.”.

It will be clear from the foregoing discussion that dendritic cells arenow proven as valuable tools in immunotherapy (particularly in thetreatment of cancer), but that DC vaccination is still at a relativelyearly stage. Methods for preparing DCs are improving continuously and anincreasing number of Phase I, II and III clinical trials are drivingintense research and development in this area. However, there is still aneed to improve efficacy at the level of DC biology.

The present inventors have now surprisingly discovered that casuarineand certain casuarine analogues have unexpected immunomodulatoryactivity, and that this activity may not be dependent on glycosidaseinhibition.

SUMMARY OF THE INVENTION

According to the invention there is provided an isolatedimmunomodulatory (e.g. immunostimulatory) polyhydroxylated pyrrolizidinecompound for use in therapy or prophylaxis having the formula:

wherein R is selected from the group comprising hydrogen, straight orbranched, unsubstituted or substituted, saturated or unsaturated acyl,alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups, or apharmaceutically acceptable salt or derivative thereof.

Preferably, the compounds of the invention are alkaloids (ashereinbefore defined).

The compound of the invention preferably has the formula:

wherein R is selected from the group comprising hydrogen, straight orbranched, unsubstituted or substituted, saturated or unsaturated acyl,alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups, or apharmaceutically acceptable salt or derivative thereof.

Particularly preferred is1R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)-1,2,6,7-tetrahydroxypyrrolizidine(casuarine), wherein R is hydrogen and which has the formula:

or a pharmaceutically acceptable derivative or salt thereof.

Particularly preferred is a casuarine glucoside, or a pharmaceuticallyacceptable salt or derivative thereof.

Other preferred compounds include 6-O-butanoylcasuarine of the formula:

or a pharmaceutically acceptable salt or derivative thereof.

A particularly preferred casuarine glucoside iscasuarine-6-α-D-glucoside of the formula:

or a pharmaceutically acceptable salt or derivative thereof.

As mentioned infra, the invention contemplates diastereomers of thecompounds of the invention. Particularly preferred are diastereomersselected from 3,7-diepisuarine (10), 7-epi-casuarine (11),3,6,7-triepi-casuarine (12), 6,7-diepi-casuarine (13) and3-epi-casuarine (14), as well as pharmaceutically acceptable salts andderivatives thereof.

Other preferred diastereomers are selected from3,7-diepi-casuarine-6-α-D-glucoside (15),7-epi-casuarine-6-α-D-glucoside (16),3,6,7-triepi-casuarine-6-α-D-glucoside (17),6,7-diepi-casuarine-6-α-D-glucoside (18) and3-epi-casuarine-6-α-D-glucoside (19), as well as pharmaceuticallyacceptable salts and derivatives thereof.

Other preferred diastereomers include 7a epimers selected from3,7,7a-triepi-casuarine, 7,7a-diepi-casuarine,3,6,7,7a-tetraepi-casuarine, 6,7,7a-triepi-casuarine and3,7a-diepi-casuarine, as well as pharmaceutically acceptable salts andderivatives thereof.

In another aspect the invention provides a method for immunomodulation(e.g. immunostimulation) comprising administering to a patient acomposition comprising a polyhydroxylated pyrrolizidine compound havingthe formula:

wherein R is selected from the group comprising hydrogen, straight orbranched, unsubstituted or substituted, saturated or unsaturated acyl,alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups, or apharmaceutically acceptable salt or derivative thereof.

The immunostimulatory methods of the invention are described in moredetail infra.

In another aspect, the invention provides a method for chemoprotectioncomprising administering the compound of the invention to a patientundergoing chemotherapy.

The invention also contemplates the use of the polyhydroxylatedpyrrolizidine compound of the invention for the manufacture of amedicament for use in immunostimulation and/or chemoprotection, as wellas a process for the manufacture of a medicament for use inimmunostimulation and/or chemoprotection, characterized in the use ofthe polyhydroxylated pyrrolizidine compound of the invention.

In another aspect, the invention contemplates a composition comprisingthe polyhydroxylated pyrrolizidine compound of the invention incombination with an immunostimulant and/or cytotoxic agent (e.g. AZT)and/or an antimicrobial (e.g. antibacterial) agent and/or an antiviralagent and/or a dendritic cell (e.g. a primed dendritic cell). Suchcompositions preferably further comprise a pharmaceutically acceptableexcipient.

In another aspect the invention contemplates a vaccine comprising thepolyhydroxylated pyrrolizidine compound of the invention in combinationwith an antigen, the compound being present in an amount sufficient toproduce an adjuvant effect on vaccination.

In another aspect the invention contemplates a pharmaceutical kit ofparts comprising the polyhydroxylated pyrrolizidine compound of theinvention in combination with an immunostimulant and/or cytotoxic agent(e.g. 5′ fluoro-uracil and ricin) and/or an antimicrobial (e.g.antibacterial) agent and/or an antiviral agent (e.g. AZT). Such kitspreferably further comprise instructions for use in immunotherapy.

The compounds of the invention have broad utility in therapy andprophylaxis, including treatments for increasing the Th1:Th2 responseratio, for example in the treatment of Th1-related diseases or disorders(e.g. proliferative disorders or microbial infection) and/or Th2-relateddiseases or disorders (for example allergies, e.g. asthma), as well asin haemorestoration, the alleviation of immunosuppression, in cytokinestimulation, in the treatment of proliferative disorders, vaccination(wherein the compound acts as an adjuvant), vaccination with dendriticcell vaccines (e.g. with primed dendritic cell vaccines, wherein thedendritic cells are contacted with the compound), in the administrationof dendritc cells in the treatment or prophylaxis of autoimmunedisorders (wherein the dendritic cells are contacted with the compound)and in wound healing. These medical uses are described in more detailbelow.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Where used herein and unless specifically indicated otherwise, thefollowing terms are intended to have the following meanings in additionto any broader (or narrower) meanings the terms might enjoy in the art:

The term adjunctive (as applied to the use of the drugs of the inventionin therapy) defines uses in which the pyrrolizidine compound isadministered together with one or more other drugs, interventions,regimens or treatments (such as surgery and/or irradiation). Suchadjunctive therapies may comprise the concurrent, separate or sequentialadministration/application of the pyrrolizidine compound of theinvention and the other treatment(s). Thus, in some embodiments,adjunctive use of the pyrrolizidine compound of the invention isreflected in the formulation of the pharmaceutical compositions of theinvention. For example, adjunctive use may be reflected in a specificunit dosage, or in formulations in which the pyrrolizidine compound ofthe invention is present in admixture with the other drug(s) with whichit is to be used adjunctively (or else physically associated with theother drug(s) within a single unit dose). In other embodiments,adjunctive use of the pyrrolizidine compound of the invention may bereflected in the composition of the pharmaceutical kits of theinvention, wherein the pyrrolizidine compound of the invention isco-packaged (e.g. as part of an array or unit doses) with the otherdrug(s) with which it is to be used adjunctively. In yet otherembodiments, adjunctive use of the pyrrolizidine compound of theinvention may be reflected in the content of the information and/orinstructions co-packaged with the pyrrolizidine compound relating toformulation and/or posology.

The term neoantigen is used herein to define any newly expressedantigenic determinant. Neoantigens may arise upon conformational changein a protein, as newly expressed determinants (especially on thesurfaces of transformed or infected cells), as the result of complexformation of one or more molecules or as the result of cleavage of amolecule with a resultant display of new antigenic determinants. Thus,as used herein, the term neoantigen covers antigens expressed uponinfection (e.g. viral infection, protozoal infection or bacterialinfection), in prion-mediated diseases (e.g. BSE and CJD), an on celltransformation (cancer), in which latter case the neoantigen may betermed a tumour-associated antigen.

The term tumour-associated antigen is used herein to define an antigenpresent in transformed (malignant or tumourous) cells which is absent(or present in lower amounts or in a different cellular compartment) innormal cells of the type from which the tumour originated. Oncogenicviruses can also induce expression of tumour antigens, which are oftenhost proteins induced by the virus.

The term herbal medicine is used herein to define a pharmaceuticalcomposition in which at least one active principle is not chemicallysynthesized and is a phytochemical constituent of a plant. In mostcases, this non-synthetic active principle is not isolated (as definedherein), but present together with other phytochemicals with which it isassociated in the source plant. In some cases, however, theplant-derived bioactive principle(s) may be in a concentrated fractionor isolated (sometimes involving high degrees of purification). In manycases, however, the herbal medicine comprises a more or less crudeextract, infusion or fraction of a plant or even an unprocessed wholeplant (or part thereof, though in such cases the plant (or plant part)is usually at least dried and/or milled.

The term bioactive principle is used herein to define a phytochemicalwhich is necessary or sufficient for the pharmaceutical efficacy of theherbal medicament in which it is comprised. In the case of the presentinvention, the bioactive principle comprises the immunomodulatorycompound of the invention (e.g. casuarine, casuarine glucoside ormixtures thereof.

The term standard specification is used herein to define acharacteristic, or a phytochemical profile, which is correlated with anacceptable quality of the herbal medicine. In this context, the termquality is used to define the overall fitness of the herbal medicamentfor its intended use, and includes the presence of one or more of thebioactive principles (at an appropriate concentration) described aboveor else the presence of one or more bioactive markers or a phytochemicalprofile which correlates with the presence of one or more of thebioactive principles (at an appropriate concentration).

The term phytochemical profile is used herein to define a set ofcharacteristics relating to different phytochemical constituents.

The term isolated as applied to the pyrrolizidine compounds of theinvention is used herein to indicate that the compound exists in aphysical milieu distinct from that in which it occurs in nature. Forexample, the isolated material may be substantially isolated (forexample purified) with respect to the complex cellular milieu in whichit naturally occurs. When the isolated material is purified, theabsolute level of purity is not critical and those skilled in the artcan readily determine appropriate levels of purity according to the useto which the material is to be put. Preferred, however, are puritylevels of 90% w/w, 99% w/w or higher. In some circumstances, theisolated compound forms part of a composition (for example a more orless crude extract containing many other substances) or buffer system,which may for example contain other components. In other circumstances,the isolated compound may be purified to essential homogeneity, forexample as determined spectrophotometrically, by NMR or bychromatography (for example GC-MS).

The term pharmaceutically acceptable derivative as applied to thepyrrolizidine compounds of the invention define compounds which areobtained (or obtainable) by chemical derivatization of the parentpyrrolizidine compounds of the invention. The pharmaceuticallyacceptable derivatives are therefore suitable for administration to oruse in contact with the tissues of humans without undue toxicity,irritation or allergic response (i.e. commensurate with a reasonablebenefit/risk ratio). Preferred derivatives are those obtained (orobtainable) by alkylation, esterification or acylation of the parentpyrrolizidine compounds of the invention. The derivatives may beimmunomodulatory per se, or may be inactive until processed in vivo. Inthe latter case, the derivatives of the invention act as pro-drugs.Particularly preferred pro-drugs are ester derivatives which areesterified at one or more of the free hydroxyls and which are activatedby hydrolysis in vivo. The pharmaceutically acceptable derivatives ofthe invention retain some or all of the immunomodulatory activity of theparent compound. In some cases, the immunomodulatory activity isincreased by derivatization. Derivatization may also augment otherbiological activities of the compound, for example bioavailabilityand/or glycosidase inhibitory activity and/or glycosidase inhibitoryprofile. For example, derivatization may increase glycosidase inhibitorypotency and/or specificity.

The term pharmaceutically acceptable salt as applied to thepyrrolizidine compounds of the invention defines any non-toxic organicor inorganic acid addition salt of the free base compounds which aresuitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and which arecommensurate with a reasonable benefit/risk ratio. Suitablepharmaceutically acceptable salts are well known in the art. Examplesare the salts with inorganic acids (for example hydrochloric,hydrobromic, sulphuric and phosphoric acids), organic carboxylic acids(for example acetic, propionic, glycolic, lactic, pyruvic, malonic,succinic, fumaric, malic, tartaric, citric, ascorbic, maleic,hydroxymaleic, dihydroxymaleic, benzoic, phenylacetic, 4-aminobenzoic,4-hydroxybenzoic, anthranilic, cinnamic, salicylic, 2-phenoxybenzoic,2-acetoxybenzoic and mandelic acid) and organic sulfonic acids (forexample methanesulfonic acid and p-toluenesulfonic acid). The drugs ofthe invention may also be converted into salts by reaction with analkali metal halide, for example sodium chloride, sodium iodide orlithium iodide. Preferably, the pyrrolizidine compounds of the inventionare converted into their salts by reaction with a stoichiometric amountof sodium chloride in the presence of a solvent such as acetone.

These salts and the free base compounds can exist in either a hydratedor a substantially anhydrous form. Crystalline forms of the compounds ofthe invention are also contemplated and in general the acid additionsalts of the pyrrolizidine compounds of the invention are crystallinematerials which are soluble in water and various hydrophilic organicsolvents and which in comparison to their free base forms, demonstratehigher melting points and an increased solubility.

In its broadest aspect, the present invention contemplates all opticalisomers, racemic forms and diastereomers of the pyrrolizidine compoundsof the invention. Those skilled in the art will appreciate that, owingto the asymmetrically substituted carbon atoms present in the compoundsof the invention, the pyrrolizidine compounds of the invention may existand be synthesised and/or isolated in optically active and racemicforms. Thus, references to the pyrrolizidine compounds of the presentinvention encompass the pyrrolizidine compounds as a mixture ofdiastereomers, as individual diastereomers, as a mixture of enantiomersas well as in the form of individual enantiomers.

Therefore, the present invention contemplates all optical isomers andracemic forms thereof of the compounds of the invention, and unlessindicated otherwise (e.g. by use of dash-wedge structural formulae) thecompounds shown herein are intended to encompass all possible opticalisomers of the compounds so depicted. In cases where the stereochemicalform of the compound is important for pharmaceutical utility, theinvention contemplates use of an isolated eutomer.

Biological Activities of the Compounds of the Invention

Without wishing to be bound by any theory, it is thought that theimmunomodulatory activity of the compounds of the invention may arisefrom the stimulation and/or suppression of cytokine secretion in vivo.In particular, it is thought that that the immunomodulatory activity ofthe compounds of the invention arises from the stimulation of secretionof one or more cytokines (e.g. one or more Th1 cytokines), includinginterleukins 2 and/or 12 (IL-2 and/or IL-12) and/or the suppression ofsecretion of one or more Th2 cytokines (e.g. IL-5).

In particular, it is thought that the immunostimulatory activity of thecompounds of the invention may arise from the stimulation of IL-12 andIL-2 by dendritic cells. This leads to the stimulation of NK cells toproduce IFN-γ and induces the development of CD4⁺ Th1 cells. The inducedTh1 cells then produce IFN-γ and IL-2. The IL-2 then enhances furtherproliferation of Th1 cells and the differentiation of pathogen (e.g.tumour and virus)-specific CD8⁺ T cells. The IL-2 also stimulates thecytolytic activity of NK cells of the innate immune system.

IL-12 is the primary mediator of type-1 immunity (the Th1 response). Itinduces natural killer (NK) cells to produce IFN-γ as part of the innateimmune response and promotes the expansion of CD4⁺ Th1 cells andcytotoxic CD8⁺ cells which produce IFN-γ. It therefore increases T-cellinvasion of tumours as well as the susceptibility of tumour cells toT-cell invasion.

Thus, the compounds of the invention are preferably stimulators ofcytokine secretion. Particularly preferred are compounds which induce,potentiate, activate or stimulate the release one or more cytokines (forexample Th1 cytokines, e.g. IL-12 and/or II-2, optionally together withone or more other cytokines) in vitro.

This primary immunomodulatory activity of the compounds of the inventionis particularly important in certain medical applications (discussed indetail infra). For example, increased production of IL-12 may overcomethe suppression of innate and cellular immunities of HIV-1-infectedindividuals and AIDS patients.

The cytokine stimulation exhibited by the compounds of the invention maybe dependent, in whole or in part, on the presence of co-stimulatoryagents. Such co-stimulatory agents may include, for example, agents thatstimulate the innate immune system, including Toll-like receptor (TLR)ligands. These ligands include microbial products such aslipopolysaccharide (LPS) and/or monophosphoryl lipid) as well as othermolecules associated with microbial infection. In many applications,such co-stimulatory agents will be present in the patient to be treatedat the time of administration of the compounds of the invention.

Without wishing to be bound by any theory, it is thought that at leastsome of the pharmacological activities of the compounds of the inventionmay be based on a secondary glycosidase inhibitory activity.

Such glycosidase inhibition may lead to any or all of the following invivo:

-   -   Modification of tumour cell glycosylation (e.g. tumour antigen        glycosylation);    -   Modification of viral protein glycosylation (e.g. virion antigen        glycosylation);    -   Modification of cell-surface protein glycosylation in infected        host cells;    -   Modification of bacterial cell walls.

This ancillary biological activity may therefore augment the primaryimmunomodulatory activity in some preferred embodiments of theinvention. It may be particularly desirable in certain medicalapplications, including the treatment of proliferative disorders (suchas cancer) or in applications where infection is attendant on immunesuppression. For example, selective modification of virion antigenglycosylation may render an infecting virus less (or non-) infectiveand/or more susceptible to endogenous immune responses. In particular,the compounds of the invention may alter the HIV viral envelopeglycoprotein gp120 glycosylation patterns, hence inhibiting the entry ofHIV into the host cell by interfering with the binding to cell surfacereceptors.

Thus, the compounds of the invention are preferably (but notnecessarily) glycosidase inhibitors. Particularly preferred arecompounds which exhibit specificity of glycosidase inhibition, forexample Glucosidase I rather than mannosidases. Such preferred compoundscan therefore be quite different in their glycosidase inhibitory profileto swainsonine and its analogues, since the latter are potent andspecific inhibitors of mannosidase.

Medical Applications of the Compounds of the Invention

The invention finds broad application in medicine, for example inmethods of therapy, prophylaxis and/or diagnosis.

These medical applications may be applied to any warm-blooded animal,including humans. The applications include veterinary applications,wherein the pyrrolizidine compounds of the invention are administered tonon-human animals, including primates, dogs, cats, horses, cattle andsheep.

The pyrrolizidine compounds of the invention are immunomodulators. Thus,they find general application in the treatment or prophylaxis ofconditions in which stimulation, augmentation or induction of the immunesystem is indicated and/or in which suppression or elimination of partor all of the immune response is indicated.

Particular medical uses of the pyrrolizidine compounds of the inventionare described in detail below. References to therapy and/or prophylaxisin the description or claims are to be interpreted accordingly and areintended to encompass inter alia the particular applications describedbelow.

1. Increasing the Th1:Th2 Response Ratio

General Considerations

As explained earlier, the immune response comprises two distinct types:the Th1 response (type-1, cellular or cell mediated immunity) and Th2response (type-2, humoral or antibody mediated immunity).

These Th1 and Th2 responses are not mutually exclusive and in manycircumstances occur in parallel. In such circumstances the balance ofthe Th1/Th2 response determines the nature (and repercussions) of theimmunological defence (as explained herein).

The Th1/Th2 balance (which can be expressed as the Th1:Th2 responseratio) is determined, at least in part, by the nature of the environment(and in particular the cytokine milieu) in which antigen priming ofnaïve helper T cells occurs when the immune system is first stimulated.

The Th1 and Th2 responses are distinguished inter alia on the basis ofcertain phenotypic changes attendant on priming and subsequentpolarization of naïve helper T cells. These phenotypic changes arecharacterized, at least in part, by the nature of the cytokines secretedby the polarized helper T cells.

Th1 cells produce so-called Th1 cytokines, which include one or more ofIL-1, TNF, IL-2, IFN-gamma, IL-12 and/or IL-18. The Th1 cytokines areinvolved in macrophage activation and Th1 cells orchestratecell-mediated defences (including cytotoxic T lymphocyte production)that form a key limb of the defence against bacterial and viral attack,as well as malignant cells.

Th2 cells produce so-called Th2 cytokines, which include one or more ofIL-4, IL-5, IL-10 and IL-13. The Th2 cytokines promote the production ofvarious antibodies and can suppress the Th1 response.

Accordingly, in the mouse, a cell that makes IFN-gamma and not IL4 isclassified as Th1, whereas a CD4⁺ cell that expresses IL-4 and notIFN-gamma is classified as Th2. Although this distinction is less clearin humans (T cells that produce only Th1 or Th2 cytokines do not appearto exist in humans), the phenotype of the T cell response (Th1 or Th2)can still be distinguished in humans on the basis of the ratio of Th1 toTh2 cytokines expressed (usually, the ratio of IFN-gamma to IL-4 and/orIL-5).

There is an increasing realization that the type of immune response isjust as important in therapy and prophylaxis as its intensity or itsduration. For example, an excess Th1 response can result in autoimmunedisease, inappropriate inflammatory responses and transplant rejection.An excess Th2 response can lead to allergies and asthma. Moreover, aperturbation in the Th1:Th2 ratio is symptomatic of many immunologicaldiseases and disorders, and the development of methods for altering theTh1:Th2 ratio is now a priority.

It has now been discovered that the immunomodulatory pyrrolizidinecompounds of the invention can increase the Th1:Th2 response ratio invivo (for example, by preferentially promoting a Th1 response and/orpreferentially suppressing a Th2 response).

Thus, the compounds of the invention find application in methods oftherapy and/or prophylaxis which comprise increasing the Th1:Th2response ratio (for example, by preferentially promoting a Th1 responseand/or preferentially suppressing a Th2 response).

The medical applications contemplated herein therefore include anydiseases, conditions or disorders in which an increase in the Th1:Th2response ratio is indicated or desired. For example, the medicalapplications contemplated include diseases, conditions or disorders inwhich stimulation of a Th1 response and/or suppression of a Th2 responseis indicated or desired.

The mechanism(s) by which the compounds of the invention increase theTh1:Th2 response ratio are not yet fully understood. It is likely thatthe activity is based, at least in part, on selective Th1 cytokineinduction (since Th1 and Th2 cytokines exhibit mutual inhibition), forexample in dendritic cells.

For example, the compounds of the invention may induce, potentiate,activate or stimulate (either directly or indirectly) the release and/oractivity (in vitro and/or in vivo) of one or more Th1 cytokines (forexample one or more cytokines selected from IFN-gamma, IL-12, IL-2 andIL-18). Particularly preferred are compounds which induce, potentiate,activate or stimulate the release and/or activity (in vitro and/or invivo) of IFN-gamma and/or IL-12 and/or IL-2.

Particularly preferred are compounds that stimulate the release of IL-2and IL-12 in dendritic cells.

The compounds of the invention may also suppress or inactivate (eitherdirectly or indirectly) the release and/or activity (in vitro and/or invivo) of one or more Th2 cytokines (for example one or more cytokinesselected from IL-4, IL-5, IL-10 and IL-13). Particularly preferred arecompounds which suppress or inactivate the release and/or activity (invitro and/or in vivo) of IL-5.

Thus, particularly preferred are compounds which exhibit a Th-1 cytokinestimulatory activity together with a complementary Th2 cytokineinhibitory activity.

Specific examples of applications falling within the general class oftreatments based on increasing the Th1:Th2 response ratio are describedin the following sections.

Th1-Related Diseases

Th1-related diseases are diseases, disorders, syndromes, conditions orinfections in which Th1 cells are involved in preventing, curing oralleviating the effects of the disease, disorder, syndrome, condition orinfection.

Th1-related diseases may also include diseases, disorders, syndromes,conditions or infections in which the Th1 component of the immuneresponse is pathologically depressed or diseases, disorders, syndromes,conditions or infections in which stimulation of a Th1 response isindicated.

Such conditions may arise, for example, from certain proliferativedisorders (typically cancers) in which the proliferating (e.g. tumour)cells exert a suppressive effect on one or more components of the Th1response. For example, tumour cells may inhibit dendritic cells, causethe expression of inhibitory receptors on T cells, down regulate MHCclass I expression and induce the secretion of anti-inflammatory factorsand immunosuppressive cytokines which deactivate or suppress immune cellcytotoxicity.

Thus, the compounds of the invention find application in the treatmentor prophylaxis of Th1-related diseases.

Examples of Th1-related diseases include infectious diseases(particularly viral infections) and proliferative disorders (e.g.cancer).

Thus, the Th1-related diseases include any malignant or pre-malignantcondition, proliferative or hyper-proliferative condition or any diseasearising or deriving from or associated with a functional or otherdisturbance or abnormality in the proliferative capacity or behaviour ofany cells or tissues of the body.

Thus, the invention finds application in the treatment or prophylaxis ofbreast cancer, colon cancer, lung cancer and prostate cancer. It alsofinds application in the treatment or prophylaxis of cancers of theblood and lymphatic systems (including Hodgkin's Disease, leukemias,lymphomas, multiple myeloma, and Waldenstrom's disease), skin cancers(including malignant melanoma), cancers of the digestive tract(including head and neck cancers, cesophageal cancer, stomach cancer,cancer of the pancreas, liver cancer, colon and rectal cancer, analcancer), cancers of the genital and urinary systems (including kidneycancer, bladder cancer, testis cancer, prostate cancer), cancers inwomen (including breast cancer, ovarian cancer, gynecological cancersand choriocarcinoma) as well as in brain, bone carcinoid,nasopharyngeal, retroperitoneal, thyroid and soft tissue tumours. Italso finds application in the treatment or prophylaxis of cancers ofunknown primary site.

The Th1-related infectious diseases include bacterial, prion (e.g. BSEand CJD), viral, fungal, protozoan and metazoan infections. For example,the Th1-related infectious diseases include infection with respiratorysyncytial virus (RSV), hepatitis B virus (HBV), Epstein-Barr, hepatitisC virus (HCV), herpes simplex type 1 and 2, herpes genitalis, herpeskeratitis, herpes encephalitis, herpes zoster, human immunodeficiencyvirus (HIV), influenza A virus, hantann virus (hemorrhagic fever), humanpapilloma virus (HPV), tuberculosis, leprosy and measles.

Particularly preferred Th1-related infectious diseases include those inwhich the pathogen occupies an intracellular compartment, includingHIV/AIDS, leishmaniasis, trypanosomiasis, influenza, tuberculosis andmalaria.

The compounds of the invention may also find application in thetreatment of patients in which the Th1 immune response is defective.Such patients may include neonates, juveniles in which the Th1 responseis immature and not fully developed, as well as older patients in whichthe Th1 response has become senescent or compromised over time. In suchpatient populations the compounds of the invention may be usedprophylactically (as a generalized type 1 immune stimulant to reduce therisks of (e.g. viral) infections.

Th2-Related Diseases and Allergy

Th2-related diseases are diseases, disorders, syndromes, conditions orinfections in which Th2 cells are implicated in (e.g. support, cause ormediate) the effects of the disease, disorder, syndrome, condition orinfection.

Thus, the compounds of the invention find application in the treatmentor prophylaxis of Th2-related diseases.

One important class of Th2-related diseases treatable with the compoundsof the invention is allergic disease.

It is well known that genetically predisposed individuals can becomesensitised (allergic) to antigens originating from a variety ofenvironmental sources. The allergic reaction occurs when a previouslysensitised individual is re-exposed to the same or to a structurallysimilar or homologous allergen. Thus, as used herein the term allergy isused to define a state of hypersensitivity induced by exposure to aparticular antigen (allergen) resulting in harmful and/or uncomfortableimmunologic reactions on subsequent exposures to the allergen.

The harmful, uncomfortable and/or undesirable immunologic reactionspresent in allergy include a wide range of symptoms. Many differentorgans and tissues may be affected, including the gastrointestinaltract, the skin, the lungs, the nose and the central nervous system. Thesymptoms may include abdominal pain, abdominal bloating, disturbance ofbowel function, vomiting, rashes, skin irritation, wheezing andshortness of breath, nasal running and nasal blockage, headache and moodchanges. In severe cases the cardiovascular and respiratory systems arecompromised and anaphylactic shock leads in extreme cases to death.

It is known that the harmful, undesirable and/or uncomfortableimmunologic reactions characteristic of allergy have a Th2 responsecomponent.

As explained above, the compounds of the invention may suppress orinactivate (either directly or indirectly) the release and/or activity(in vitro and/or in vivo) of one or more Th2 cytokines (for example oneor more cytokines selected from IL-4, IL-5, IL-10 and IL-13). Thus, thecompounds of the invention may be used to effect a remedial orpalliative modulation of the harmful and/or uncomfortable immunologicreactions characteristic of allergic reactions by inhibiting,suppressing or eliminating the Th2 response to the allergen.

The compounds of the invention therefore find application in thetreatment or prophylaxis of allergy.

Any allergy may be treated according to the invention, including atopicallergy, allergic rhinitis, allergic conjunctivitis, atopic dermatitis,hypereosinophilia, irritable bowel syndrome, allergen-induced migraine,bacterial allergy, bronchial allergy (asthma), contact allergy(dermatitis), delayed allergy, pollen allergy (hay fever), drug allergy,sting allergy, bite allergy, gastrointestinal or food allergy (includingthat associated with inflammatory bowel disease, including ulcerativecolitis and Crohn's disease) and physical allergy. Physical allergiesinclude cold allergy (cold urticaria or angioedema), heat allergy(cholinergic urticaria) and photosensitivity.

Particularly important is the treatment or prophylaxis of asthma.

2. Haemorestoration

The pyrrolizidine compounds of the invention increase splenic and bonemarrow cell proliferation and can act as myeloproliferative agents. Theytherefore find application as haemorestoratives.

Haemorestoration may be indicated following immunosuppressant therapies(such as cyclosporine A, azathioprine or immunosuppressantradiotherapies), chemotherapy (including treatment with bothcycle-specific and non-specific chemotherapeutic agents), steroidadministration or other forms of surgical or medical intervention(including radiotherapy). Thus, the use of the pyrrolizidine compoundsof the invention as haemorestoratives may be adjunctive to othertreatments which tend to depress splenic and bone marrow cellpopulations. Particularly preferred adjunctive therapies according tothe invention include the administration of an immunorestorative dose ofthe pyrrolizidine compound of the invention adjunctive to: (a)chemotherapy; and/or (b) radiotherapy; and/or (c) bone marrowtransplantation; and/or (d) haemoablative immunotherapy.

3. Alleviation of Immunosuppression

The pyrrolizidine compounds of the invention may be used to alleviate,control or modify states in which the immune system is partially orcompletely suppressed or depressed. Such states may arise fromcongenital (inherited) conditions, be acquired (e.g. by infection ormalignancy) or induced (e.g. deliberately as part of the management oftransplants or cancers).

Thus, the pyrrolizidine compounds of the invention may find applicationas adjunctive immunomodulators (e.g. immunostimulants) in the treatmentand/or management of various diseases (including certain cancers) ormedical interventions (including radiotherapy, immunosuppressant therapy(such as the administration of cyclosporine A, azathioprine orimmunosuppressant radiotherapies), chemotherapy and cytotoxic drugadministration (for example the administration of ricin,cyclophosphamide, cortisone acetate, vinblastine, vincristine,adriamycin, mercaptopurine, 5-fluorouracil, mitomycin C, chloramphenicoland other steroid-based therapies). They may therefore be used aschemoprotectants in the management of various cancers and infections(including bacterial and viral infections, e.g. HIV infection) or toinduce appropriate and complementary immunotherapeutic activity duringconventional immunotherapy.

In particular, the pyrrolizidine compounds of the invention may findapplication as immunostimulants in the treatment or management ofmicrobial infections which are associated with immune-suppressed states,including many viral infections (including HIV infection in AIDS) and inother situations where a patient has been immunocompromised (e.g.following infection with hepatitis C, or other viruses or infectiousagents including bacteria, fungi, and parasites, in patients undergoingbone marrow transplants, and in patients with chemical or tumor-inducedimmune suppression).

Other diseases or disorders which may give rise to an immunosuppressedstate treatable according to the invention include:ataxia-telangiectasia; DiGeorge syndrome; Chediak-Higashi syndrome; Jobsyndrome; leukocyte adhesion defects; panhypogammaglobulinemia (e.g.associated with Bruton disease or congenital agammaglobulinemia);selective deficiency of IgA; combined immunodeficiency disease;Wiscott-Aldrich syndrome and complement deficiencies. It may beassociated with organ and/or tissue (e.g. bone marrow) transplantationor grafting, in which applications the pyrrolizidine compounds of theinvention may be used adjunctively as part of an overall treatmentregimen including surgery and post-operative management of immunestatus.

4. Cytokine Stimulation

The pyrrolizidine compounds of the invention may be used to induce,potentiate or activate various cytokines in vivo, including variousinterleukins (including IL-2 and/or IL-1).

Accordingly, the pyrrolizidine compounds of the invention find generalapplication in the treatment or prophylaxis of conditions in which thein vivo induction, potentiation or activation of one or more cytokines(e.g. IL-12 and/or II-2) is indicated. Such applications may be employedto stimulate particular elements of the cellular immunity system,including dendritic cells, macrophages (e.g. tissue-specificmacrophages), CTL, NK, NKT, B and LAK cells.

In such applications, the compounds of the invention may be employed asan adjunct to gene therapies designed to increase the production ofendogenous cytokines (for example IL-2).

5. Treatment of Proliferative Disorders

The invention finds application in the treatment of proliferativedisorders, including various cancers and cancer metastasis. For example,the pyrrolizidine compounds of the invention may find particularapplication in the treatment of leukemias, lymphomas, melanomas,adenomas, sarcomas, carcinomas of solid tissues, melanoma (includingmelanoma of the eye), pancreatic cancer, cervico-uterine cancer, cancersof the kidney, stomach, lung, ovary, rectum, breast, prostate, bowel,gastric, liver, thyroid, neck, cervix, salivary gland, leg, tongue, lip,bile duct, pelvis, mediastinum, urethra, lung, bladder, esophagus andcolon, and Kaposi's Sarcoma (e.g. when associated with AIDS).

In such applications the compounds of the invention may exhibit asecondary glycosidase inhibitory activity.

The invention may therefore find application in methods of therapy orprophylaxis which comprise the modification of tumour cell glycosylation(e.g. tumour antigen glycosylation), the modification of viral proteinglycosylation (e.g. virion antigen glycosylation), the modification ofcell-surface protein glycosylation in infected host cells and/or themodification of bacterial cell walls, hence promoting an increasedimmune response or inhibiting growth/infectivity directly.

6. Use as Adjuvant

The pyrrolizidine compounds of the invention find utility as vaccineadjuvants, in which embodiments they may promote, induce or enhance animmune response to antigens, particularly antigens having low intrinsicimmunogenicity. Without wishing to be bound by any theory, thepyrrolizidine compounds of the invention may augment vaccineimmunogenicity by stimulating cytokine release, thereby promoting T-cellhelp for B cell and CTL responses. They may also change glycosylation ofcancer or viral antigens and increase vaccine effectiveness.

When used as adjuvant, the compounds of the invention may beadministered concurrently, separately or sequentially withadministration of the vaccine. The invention finds application in anyvaccine, but may be particularly as a subunit vaccine, a conjugatevaccine, a DNA vaccine, a recombinant vaccine or a mucosal vaccine. Thevaccine may be therapeutic or prophylactic. It may be usedimmunoprophylactically or immunotherapeutically in both human andnon-human subjects. Preferred non-human subjects include mammals andbirds. Particularly preferred are veterinary applications. Suchapplications include the treatment or prophylaxis of infection indomesticated animals (for example dogs and cats) and livestock (e.g.sheep, cows, pigs, horses, chickens and turkeys).

Thus, in some embodiments, the pyrrolizidine compound of the inventionmay be present in admixture with other vaccine component(s), or elseco-packaged (e.g. as part of an array of unit doses) with the othervaccine components with which it is to be used as adjuvant. In yet otherembodiments, the use of the pyrrolizidine compounds of the invention asadjuvant is simply reflected in the content of the information and/orinstructions co-packaged with the vaccine components and relating to thevaccination procedure, vaccine formulation and/or posology.

7. Dendritic Cell-Based Applications

As described above, it has now been found that the pyrrolizidinecompounds of the invention may induce sustained and pronounced cytokineproduction (e.g. sustained and pronounced IL-12 and/or IL-2 production)in dendritc cells. Thus, the compounds of the invention find applicationin methods of therapy or prophylaxis comprising the induction ofcytokine production in dendritic cells or in which the induction ofcytokine production in dendritic cells is indicated or required.

Dendritic Cell Vaccines

In one dendritic cell-based treatment paradigm, the cells are pulsed(primed or spiked) with a particular antigen or antigens (for example,tumour antigen(s)) and then administered to promote a Th1 immuneresponse. The responding T cells include helper cells, especially Th1CD4⁺ cells (which produce IFN-γ) and killer cells (especially CD8⁺cytolytic T lymphocytes). The dendritc cells may also mediate responsesby other classes of lymphocytes (B, NK, and NKT cells). They may alsoelicit T cell memory, a critical goal of vaccination.

With regard to antigen selection for use in the dendritic cell vaccinesof the invention, both defined and undefined antigens can be employed.The antigens can be xenoantigens or autoantigens. One or more definedneoantigen(s) may be selected: in the case of cancer treatment, theneoantigen(s) may comprise a tumour-associated antigen.

However, most preferred for use according to the invention are peptides(for example, synthetic 9-11 amino acid peptides) containing definedantigens. Such peptides may comprise natural sequences. Alternatively,they may be synthetic analogues designed for enhanced MHC binding.

In other embodiments, the antigens used according to the invention areprovided in the form of immune complexes. These are preferably deliveredto Fc-receptor-bearing DCs so that both MHC class I and MHC class IIpeptide sequences are formed. In this way, dendritic cell vaccines canbe used according to the invention for inducing both CTLs and Th cells.

In another approach to antigen selection for use according to theinvention, the whole antigenic repertoire of any given tumour (or othertarget cell, such as a virally-infected cell) is explored. Thus, inanother embodiment of the invention there is provided DC-tumour cellhybrids in which the dendritic cells are treated with compound (therebyto induce the expression of IL2) before or after hybridisation.

In yet other embodiments, necrotic or apoptotic tumour cells or celllysates (for example lysates of infected cells or tumour cells) areused.

Antigens derived from fresh tumour cells (rather than tumour cell linesor defined antigens) may also be employed.

It is also contemplated that the compounds of the invention beincorporated into cellular antigens by introducing them into thecellular membrane or into an intracellular compartment (as described forexample in WO96017614, the contents of which are incorporated herein byreference).

Various techniques can be used to deliver the selected antigen(s) to theDCs (variously referred to in the art as antigen loading, pulsing,priming or spiking). Preferred are loading techniques which load the DCsinternally: this can be achieved through the use of peptides linked tocell-penetrating moieties.

Antigens can also be loaded by transfecting the DCs with encodingnucleic acid (e.g. by electroporation) such that the antigens areexpressed by the DC, processed and presented at the cell surface. Thisapproach avoids the need for expensive GMP proteins and antibodies. RNAis preferred for this purpose, since it produces only transientexpression (albeit sufficient for antigen processing) and avoids thepotential problems associated with the integration of DNA and attendantlong-term expression/mutagenesis. Such transfection techniques alsopermit exploration of the whole antigenic repertoire of a target cell byuse of total or PCR-amplified tumour RNA.

Current strategies for using dendritic cells in this way focus onidentifying specific tumour antigens and defining antigenic peptidesthat bind to the particular MHC alleles expressed by each patient.However, a more general approach would involve the stimulation of thedendritic cells in a manner appropriate for potentiating Th1 responsesirrespective of the antigens present and either with or without antigenpriming. Cytokine production by activated dendritic cells would thenpromote the appropriate Th1 response.

The dendritic cell based vaccines of the invention find particularapplication in the treatment or prophylaxis of various proliferativedisorders (including various cancers, as described below). In suchapplications, the dendritic cells are preferably pulsed (primed orspiked) with one or more tumour antigens ex vivo and the compounds ofthe invention used to potentiate the dendritic cell component of thevaccine by contacting the dendritic cells with the compound either exvivo (before or after pulsing of the cells) or in vivo (for example byco-administration, either concurrently, separately or sequentially, ofthe dendritic cells and the compound).

The dendritic cell based vaccines of the invention may be used in thetreatment or prophylaxis of any malignant or pre-malignant condition,proliferative or hyper-proliferative condition or any disease arising orderiving from or associated with a functional or other disturbance orabnormality in the proliferative capacity or behaviour of any cells ortissues of the body.

Thus, the invention finds application in the treatment or prophylaxis ofbreast cancer, colon cancer, lung cancer and prostate cancer. It alsofinds application in the treatment or prophylaxis of cancers of theblood and lymphatic systems (including Hodgkin's Disease, leukemias,lymphomas, multiple myeloma, and Waldenstrom's disease), skin cancers(including malignant melanoma), cancers of the digestive tract(including head and neck cancers, cesophageal cancer, stomach cancer,cancer of the pancreas, liver cancer, colon and rectal cancer, analcancer), cancers of the genital and urinary systems (including kidneycancer, bladder cancer, testis cancer, prostate cancer), cancers inwomen (including breast cancer, ovarian cancer, gynecological cancersand choriocarcinoma) as well as in brain, bone carcinoid,nasopharyngeal, retroperitoneal, thyroid and soft tissue tumours. Italso finds application in the treatment or prophylaxis of cancers ofunknown primary site.

The dendritic cell based vaccines of the invention also find applicationin the treatment or prophylaxis of various infections, includingbacterial, viral, fungal, protozoan and metazoan infections. Forexample, the vaccines may be used in the treatment or prophylaxis ofinfection with respiratory syncytial virus (RSV), Epstein-Barr,hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex type 1and 2, herpes genitalis, herpes keratitis, herpes encephalitis, herpeszoster, human immunodeficiency virus (HIV), influenza A virus, hantannvirus (hemorrhagic fever), human papilloma virus (HPV), tuberculosis,leprosy and measles.

Particularly preferred is the treatment or prophylaxis of infections inwhich the pathogen occupies an intracellular compartment or causes theexpression of neoantigens by host cells, including HIV/AIDS, leishmania,trypanosomiasis, influenza, tuberculosis and malaria.

The present invention also contemplates a more general approach to DCcell-based therapy which involves the stimulation of the dendritc cellswith the compound of the invention irrespective of the antigens presentand either with or without antigen priming.

Thus, the invention finds application in therapies in which dendriticcells exposed to the compound of the invention are targeted to diseasedor infected tissue (for example injected directly into a tumour), wherethe cells can prime endogenous T cells extranodally. In suchembodiments, the invention contemplates targeting of DCs to a tumour andtheir activation in situ to elicit immune responses without the need forex vivo antigen loading.

In yet another embodiment, the invention contemplates in situ DCvaccination where antigen is targeted to DCs in vivo which are thenexpanded and induced to mature in situ (by the co-administration of oneor more DC maturation stimulants). In such embodiments, antigen istargeted to endogenous DCs by any convenient method, for example throughthe use of exosomes (as described in Thery et al. (2002) Nat Rev Immunol2: 569-579).

Any class of dendritic cell may be used according to the invention.Thus, the dendritic cells may be myeloid or lymphoid, or mixturesthereof. The myeloid dendritic cells, if used, may be of the Langerhanscell type or interstitial DCs. Alternatively, mixtures of these myeloidsubsets may be used. Especially preferred is the use of monocyte-derivedDCs (Mo-DCs).

Helper proteins may be used to potentiate the activity of the dendriticcell vaccines of the invention.

Dendritic Cell-Based Approaches to Autoimmune Disorders

Dendritic cells are also involved in regulating and maintainingimmunological tolerance: in the absence of maturation, the cells induceantigen-specific silencing or tolerance. Thus, in another dendriticcell-based treatment paradigm the cells are administered as part of animmunomodulatory intervention designed to combat autoimmune disorders.

In such applications, the suppressive potential of dendritic cells hasbeen enhanced by in vitro transfection with genes encoding cytokines.However, such gene therapy approaches are inherently dangerous and amore efficient and attractive approach would be to pulse dendritic cellsin vitro with biologically active compounds which stimulate anappropriate cytokine secretion pattern in the dendritic cells.

As described above, it has now been discovered that the pyrrolizidinecompounds of the invention can induce sustained and pronounced cytokineproduction in dendritic cells. Thus, the compounds of the invention findapplication in the enhancement of the suppressive potential of dendriticcells.

Thus, the invention finds application in the treatment or prophylaxis ofautoimmune disorders, including myasthenia gravis, rheumatoid arthritis,systemic lupus erythematosus, Sjogren syndrome, scleroderma,polymyositis and dermomyositis, ankylosing spondylitis, and rheumaticfever, insulin-dependent diabetes, thyroid diseases (including Grave'sdisease and Hashimoto thyroidits), Addison's disease, multiplesclerosis, psoriasis, inflammatory bowel disease, ulcerative colitis andautoimmune male and female infertility.

8. Wound Healing

The pyrrolizidine compounds of the invention can reverse a Th2 typesplenocyte response ex vivo in a normally non-healing infectious diseasemodel. Antigen specific splenocyte IFN-gamma can be significantlyincreased and IL-5 production significantly reduced in such models,indicative of a healing response.

Thus, the invention finds application in the treatment of wounds. Inparticular, the invention finds application in the treatment orprophylaxis of wounds and lesions, for example those associated withpost-operative healing, burns, infection (e.g. necrotic lesions),malignancy or trauma (e.g. associated with cardiovascular disorders suchas stroke or induced as part of a surgical intervention).

The wound treatments may involve the selective suppression orelimination of a Th2 response (for example to eliminate or suppress aninappropriate or harmful inflammatory response).

Posology

The pyrrolizidine compounds of the present invention can be administeredby oral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration.

The amount of the pyrrolizidine compound administered can vary widelyaccording to the particular dosage unit employed, the period oftreatment, the age and sex of the patient treated, the nature and extentof the disorder treated, and the particular pyrrolizidine compoundselected.

Moreover, the pyrrolizidine compounds of the invention can be used inconjunction with other agents known to be useful in the treatment ofdiseases, disorders or infections where immunostimulation is indicated(as described infra) and in such embodiments the dose may be adjustedaccordingly.

In general, the effective amount of the pyrrolizidine compoundadministered will generally range from about 0.01 mg/kg to 500 mg/kgdaily. A unit dosage may contain from 0.05 to 500 mg of thepyrrolizidine compound, and can be taken one or more times per day. Thepyrrolizidine compound can be administered with a pharmaceutical carrierusing conventional dosage unit forms either orally, parenterally, ortopically, as described below.

The preferred route of administration is oral administration. In generala suitable dose will be in the range of 0.01 to 500 mg per kilogram bodyweight of the recipient per day, preferably in the range of 0.1 to 50 mgper kilogram body weight per day and most preferably in the range 1 to 5mg per kilogram body weight per day.

The desired dose is preferably presented as a single dose for dailyadministration. However, two, three, four, five or six or more sub-dosesadministered at appropriate intervals throughout the day may also beemployed.

These sub-doses may be administered in unit dosage forms, for example,containing 0.001 to 100 mg, preferably 0.01 to 10 mg, and mostpreferably 0.5 to 1.0 mg of active ingredient per unit dosage form.

Formulation

The compositions of the invention comprise the pyrrolizidine compound ofthe invention, optionally together with a pharmaceutically acceptableexcipient.

The pyrrolizidine compound of the invention may take any form. It may besynthetic, purified or isolated from natural sources (for example fromCasuarina equisetifolia or Eugenia jambolana), using techniquesdescribed in the art (and referenced infra).

When isolated from a natural source, the pyrrolizidine compound of theinvention may be purified. However, the compositions of the inventionmay take the form of herbal medicines, as hereinbefore defined. Suchherbal medicines preferably are analysed to determine whether they meeta standard specification prior to use.

The herbal medicines for use according to the invention may be driedplant material. Alternatively, the herbal medicine may be processedplant material, the processing involving physical or chemicalpre-processing, for example powdering, grinding, freezing, evaporation,filtration, pressing, spray drying, extrusion, supercritical solventextraction and tincture production. In cases where the herbal medicineis administered or sold in the form of a whole plant (or part thereof),the plant material may be dried prior to use. Any convenient form ofdrying may be used, including freeze-drying, spray drying or air-drying.

In embodiments where the pyrrolizidine compound of the invention isformulated together with a pharmaceutically acceptable excipient, anysuitable excipient may be used, including for example inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavouring agents, colouring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc.

The pharmaceutical compositions may take any suitable form, and includefor example tablets, elixirs, capsules, solutions, suspensions, powders,granules and aerosols.

The pharmaceutical composition may take the form of a kit of parts,which kit may comprise the composition of the invention together withinstructions for use and/or a plurality of different components in unitdosage form.

Tablets for oral use may include the pyrrolizidine compound of theinvention, either alone or together with other plant material associatedwith the botanical source(s) (in the case of herbal medicineembodiments). The tablets may contain the pyrrolizidine compound of theinvention mixed with pharmaceutically acceptable excipients, such asinert diluents, disintegrating agents, binding agents, lubricatingagents, sweetening agents, flavouring agents, colouring agents andpreservatives. Suitable inert diluents include sodium and calciumcarbonate, sodium and calcium phosphate, and lactose, while corn starchand alginic acid are suitable disintegrating agents. Binding agents mayinclude starch and gelatin, while the lubricating agent, if present,will generally be magnesium stearate, stearic acid or talc. If desired,the tablets may be coated with a material such as glyceryl monostearateor glyceryl distearate, to delay absorption in the gastrointestinaltract

Capsules for oral use include hard gelatin capsules in which thepyrrolizidine compound of the invention is mixed with a solid diluent,and soft gelatin capsules wherein the active ingredient is mixed withwater or an oil such as peanut oil, liquid paraffin or olive oil.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the compounds of the invention will generally be provided in sterileaqueous solutions or suspensions, buffered to an appropriate pH andisotonicity.

Suitable aqueous vehicles include Ringer's solution and isotonic sodiumchloride. Aqueous suspensions according to the invention may includesuspending agents such as cellulose derivatives, sodium alginate,polyvinylpyrrolidone and gum tragacanth, and a wetting agent such aslecithin. Suitable preservatives for aqueous suspensions include ethyland n-propyl p-hydroxybenzoate.

The compounds of the invention may also be presented as liposomeformulations.

For oral administration the pyrrolizidine compound of the invention canbe formulated into solid or liquid preparations such as capsules, pills,tablets, troches, lozenges, melts, powders, granules, solutions,suspensions, dispersions or emulsions (which solutions, suspensionsdispersions or emulsions may be aqueous or non-aqueous). The solid unitdosage forms can be a capsule which can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers such as lactose, sucrose, calciumphosphate, and cornstarch.

In another embodiment, the pyrrolizidine compounds of the invention aretableted with conventional tablet bases such as lactose, sucrose, andcornstarch in combination with binders such as acacia, cornstarch, orgelatin, disintegrating agents intended to assist the break-up anddissolution of the tablet following administration such as potatostarch, alginic acid, corn starch, and guar gum, lubricants intended toimprove the flow of tablet granulations and to prevent the adhesion oftablet material to the surfaces of the tablet dies and punches, forexample, talc, stearic acid, or magnesium, calcium, or zinc stearate,dyes, coloring agents, and flavoring agents intended to enhance theaesthetic qualities of the tablets and make them more acceptable to thepatient.

Suitable excipients for use in oral liquid dosage forms include diluentssuch as water and alcohols, for example, ethanol, benzyl alcohol, andthe polyethylene alcohols, either with or without the addition of apharmaceutically acceptably surfactant, suspending agent or emulsifyingagent.

The pyrrolizidine compounds of the invention may also be administeredparenterally, that is, subcutaneously, intravenously, intramuscularly,or interperitoneally.

In such embodiments, the pyrrolizidine compound is provided asinjectable doses in a physiologically acceptable diluent together with apharmaceutical carrier (which can be a sterile liquid or mixture ofliquids). Suitable liquids include water, saline, aqueous dextrose andrelated sugar solutions, an alcohol (such as ethanol, isopropanol, orhexadecyl alcohol), glycols (such as propylene glycol or polyethyleneglycol), glycerol ketals (such as2,2-dimethyl-1,3-dioxolane-4-methanol), ethers (such aspoly(ethylene-glycol) 400), an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant (such as a soap ora detergent), suspending agent (such as pectin, carnomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose), or emulsifying agent and other pharmaceuticallyadjuvants. Suitable oils which can be used in the parenteralformulations of this invention are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineraloil.

Suitable fatty acids include oleic acid, stearic acid, and isostearicacid. Suitable fatty acid esters are, for example, ethyl oleate andisopropyl myristate.

Suitable soaps include fatty alkali metal, ammonium, and triethanolaminesalts and suitable detergents include cationic detergents, for example,dimethyl dialkyl ammonium halides, alkyl pyridinium halides, andalkylamines acetates; anionic detergents, for example, alkyl, aryl, andolefin sulphonates, alkyl, olefin, ether, and monoglyceride sulphates,and sulphosuccinates; nonionic detergents, for example, fatty amineoxides, fatty acid alkanolamides, and polyoxyethylenepolypropylenecopolymers; and amphoteric detergents, for example,alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammoniumsalts, as well as mixtures.

The parenteral compositions of this invention will typically containfrom about 0.5 to about 25% by weight of the pyrrolizidine compound ofthe invention in solution. Preservatives and buffers may also be used.In order to minimize or eliminate irritation at the site of injection,such compositions may contain a non-ionic surfactant having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5 to about15% by weight. The surfactant can be a single component having the aboveHLB or can be a mixture of two or more components having the desiredHLB. Illustrative of surfactants used in parenteral formulations are theclass of polyethylene sorbitan fatty acid esters, for example, sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol.

The pyrrolizidine compounds of the invention may also be administeredtopically, and when done so the carrier may suitably comprise asolution, ointment or gel base. The base, for example, may comprise oneor more of the following: petrolatum, lanolin, polyethylene glycols, beewax, mineral oil, diluents such as water and alcohol, and emulsifiersand stabilizers. Topical formulations may contain a concentration of thecompound from about 0.1 to about 10% w/v (weight per unit volume).

When used adjunctively, the pyrrolizidine compounds of the invention maybe formulated for use with one or more other drug(s), in particular, thepyrrolizidine compounds of the invention may be used in combination withantitumor agents, antimicrobial agents, anti-inflammatories,antiproliferative agents and/or other immunomodulatory (e.g.immunostimulatory) agents. For example, the pyrrolizidine compounds ofthe invention may be used with anti-viral and/or anti-proliferativeagents such as cytokines, including interleukins-2 and 12, interferonsand inducers thereof, tumor necrosis factor (TNF) and/or transforminggrowth factor (TGF), as well as with myelosuppressive agents and/orchemotherapeutic agents (such as doxorubicin, 5-fluorouracil,cyclophosphamide and methotrexate), isoniazid (e.g. in the prevention ortreatment of peripheral neuropathy) and with analgesics (e.g. NSAIDs)for the prevention and treatment of gastroduodenal ulcers.

Thus, adjunctive use may be reflected in a specific unit dosage designedto be compatible (or to synergize) with the other drug(s), or informulations in which the pyrrolizidine compound is admixed with one ormore antitumor agents, antimicrobial agents and/or antiinflammatories(or else physically associated with the other drug(s) within a singleunit dose). Adjunctive uses may also be reflected in the composition ofthe pharmaceutical kits of the invention, in which the pyrrolizidinecompound of the invention is co-packaged (e.g. as part of an array ofunit doses) with the antitumor agents, antimicrobial agents and/orantiinflammatories. Adjunctive use may also be reflected in informationand/or instructions relating to the co-administration of thepyrrolizidine compound with antitumor agents, antimicrobial agentsand/or antiinflammatories.

EXEMPLIFICATION

The invention will now be described with reference to specific Examples.These are merely exemplary and for illustrative purposes only: they arenot intended to be limiting in any way to the scope of the monopolyclaimed or to the invention described. These examples constitute thebest mode currently contemplated for practicing the invention.

Example 1 Induction of IL-12 Secretion in Dendritic Cells

Mice

BALB/c male and female mice bred and maintained at the University ofStrathclyde under conventional conditions were used at 8 weeks old.

Isolation of Bone Marrow and Culture of Dendritic Cells

Bone marrow was obtained from the femurs of mice. The femurs were washedin 70% ethanol and placed in a 40 clean petri dish. Dendritic cell (DC)medium (2.5% granulocyte-macrophage colony-stimulating factor (GM-CSF),10% heat and activated foetal calf serum, 1% L-glutamine, 1%Penicillin/Streptomycin in RPMI-1640 medium) was injected into the bonemarrow of the femur by a pumping action and the cells and medium werecollected. 1 ml of the cells in medium was added to a 75 cm² flask with15 mls of DC medium. The flasks were then incubated at 37° C., 5% CO₂ toallow DC growth and development. After 5 days an additional 10 mls of DCmedium was added.

Harvesting of Dendritic Cells

After 10 days of incubation of bone marrow with GM-CSF, the dendriticcells were harvested. This process was carried out in a tissue culturehood. The contents of the flasks were poured into centrifuge tubes toensure collection of floating DCs. Approximately 10 mls of cooledphosphate buffered saline (PBS) was added to each empty flask, theflasks gently agitated and the contents collected. This ensured recoveryof adhesive DCs. The collected contents of the flasks were centrifugedfor 5 minutes at 200 g and the pellet resuspended in 2 mls of DC mediumwithout GM-CSF. A cell count was then carried out.

Cell Count and Assay Conditions

Cells were counted using a haemocytometer. Approximately 20 μl of theresuspended cells was pipetted into the chamber of the haemocytometer,the cells were adjusted to the correct cell concentration (approx. 5×10⁴and not less than 1×10⁴, per well) and then plated out for assay.

The plates were incubated overnight at 37° C. with 5% CO₂ and allowed tosettle (harvesting stimulates them). The next day the compounds (50μg/ml and 20 μg/ml) and controls were added then again incubated at 37°C. with 5% CO₂ for 24 hrs (or 48 hrs). Harvesting and addition of thecompounds was all done in a hood. The plates were then frozen to killthe cells and once defrosted the supernatant analysed as describedbelow.

Measurement of IL-12

Using an enzyme linked immunosorbent assay (ELISA) IL-12 concentrationin the supernatants was measured. All reagents used in this assay werefrom PharMingen. A 96-well flat-bottomed ELISA plate was coated withpurified rat anti-mouse IL-12 (p40/p70) MAb (Cat no. 554478) at 2 μg/mldiluted in PBS pH 9.0 at 50 μl/well. The plate was then covered in clingfilm and incubated at 4° C. Following incubation the plate was washed 3times in washing buffer and dried. 200 μl of blocking buffer (10% foetalcalf serum in PBS pH 7.0) was added to each well then covered in clingfilm and incubated at 37° C. for 45 minutes. The plate was washed 3times and dried. Recombinant mouse II-12 standard was added at 30 μl induplicate wells, starting at 10 ng/ml then 5, 2.5, 1.25, 0.625, 0.31,0.156, 0.078, 0.039, 0.020, 0.010, 0.005 ng/ml. Standards were dilutedin blocking buffer. The supernatant samples were added in at 50 μl/well.The plate was then covered in cling film and incubated for 2 hours at37° C. The plate was then washed 4 times, dried and the secondaryantibody added.

Biotin labeled anti-mouse IL-12 (p40/p70) MAb (Cat no. 18482D) at 1μg/ml (diluted in blocking buffer) was added to each well at a volume of100 μl/well. The plate was covered in cling film and incubated at 37° C.for 1 hour. The plate was then washed 5 times, dried and the conjugateadded. Streptavidin-AKP (Cat no. 13043E) at 100 μl/well was added at adilution of 1/2000 in blocking buffer followed by incubation under clingfilm at 37° C. for 45 minutes.

The plate was finally washed 6 times, dried and the substrate added:pNPP (Sigma) in glycine buffer at 1 mg/ml was added at 100 μl/well. Theplate was then covered in tinfoil, incubated at 37° C. and checked every30 minutes for a colour change.

The plate was then read at 405 nm using a SPECTRAmax 190 spectrometer.The results are shown in FIGS. 1 and 2, in which LPS islipopolysaccharide, IFN-g is interferon gamma, 462a is casuarine (8),462b is casuarine-6-α-D-glucopyranose (9), 23 is 7-epicasuarine (11) and24 is 3,7-diepi-casuarine (10).

When tested at 50 μg/ml in the same assay, swainsonine (4) failed toinduce IL-12 secretion. Similar studies with other compounds forcomparative purposes are shown in Table 1.1, below. COM- IL-12 POUNDSTRUCTURE RELEASE casuarine (8)

Yes casuarine- 6-α- D- gluco- pyranose (9)

Yes 3,7-diepi- casuarine (10)

Yes 7-epi- casuarine (11)

Yes 3-epi- casuarine (14)

Yes Castano- spermine (20)

No Swain- sonine (4)

No 1-Deoxy- no- jirimycin (DNJ) (21)

No 7- epialexine (22)

No 3,7a- diepi- alexine (23)

No Alexine (1)

No

Example 2 Stimulation of IL-2 Production by Dendritic Cells

The protocols described in Example 1 above were carried out but theappropriate Mabs and standards for determination of II-2 weresubstituted. The results are shown in Table 2.1, below. Treatment IL-2(units/ml) LPS 0.00 LPS + IFN-_(Y) 0.00 3,7-diepi-casuarine (10) 0.003,7-diepi-casuarine (10) + LPS 0.69

Example 3 Cytokine Modulation in Spleen Cells

Mice

BALB/c male and female mice bred and maintained at the University ofStrathclyde under conventional conditions were used at varying age.

Isolation of Spleen Cells and Culture of Spleen Cells

The mouse spleen was removed aseptically and placed in a sterile petridish containing 5 mls of complete medium (RPMI, 1% L-Glutamine, 1%Penicillin/Streptomycin and 10% foetal calf serum). Cells suspensionswere prepared by using the end of a syringe and grinding the spleenthrough a wire mesh. The cell suspension was then centrifuged at 1000rpm for 5 minutes. To remove the erythrocytes, the cell pellet wasresuspended in Boyle's solution (Tris 0.17M Ammonium Chloride 0.16M) andcentrifuged again for 5 minutes. The pellet was then washed in medium afurther two times, then resuspended in 3 mls medium. A cell count wasthen carried out.

Experimental Protocol

All spleen cell experiments were carried out in 96-well tissue cultureplates. 100 μl aliquots of 5×10⁵/well cells were added to all wells andeach well had a final volume of 200 μl. Unstimulated wells contained 100μl of cells and 100 μl of medium. The stimulated wells contained 100 μlof cells plus 50 μl of LPS at 1 μg/ml or 50 μl anti-CD3 at 0.5 μg/ml and50 μl of medium. The remaining wells contained 100 μl cells, 50 μl ofMNLP compound and either 50 μl of anti-CD3 or medium alone.

Measurement of IL-12, IL-2, IL-5 and IFN-γ

The appropriate Mabs and standards were used according to the protocoldescribed for IL-12 (described in Example 1, above). The results areshown in Tables 3.1-3.3, below. TABLE 3.1 Promotion of activatedsplenocyte (T-cell) IFN-_(Y) production Treatment IFN-_(Y (ng/ml)) None(control) 0.64 αCD3 3.21 3,7-diepi-casuarine (10) 0.223,7-diepi-casuarine (10) + αCD3 13.50

TABLE 3.2 Effect of castanospermine on splenocyte IFN-y productionTreatment IFN-_(Y (ng/ml)) None (control) <1.0 αCD3 22.5 Castanospermine(20) <1.0 Castanospermine (20) + aCD3 9.0

As can be seen from the results shown in Tables 3.1 and 3.2, compoundsaccording to the invention stimulate IFN-γ secretion/production insplenocytes, whereas castanospermine inhibits the production of thiscytokine in such assays. Similar tests carried out with1-Deoxynojirimycin (DNJ) (21) showed that this imino sugar alsoinhibited IFN-γ secretion/production in splenocytes (data not shown).

Example 4 Inhibition of Glycosidase Activity

All enzymes were purchased from Sigma, as were the appropriatep-nitrophenyl substrates. Assays were carried out in microtitre plates.Enzymes were assayed in 0.1M citric acid/0.2M di-sodium hydrogenphosphate (McIlvaine) buffers at the optimum pH for the enzyme. Allassays were carried out at −20° C. For screening assays the incubationassay consisted of 10 μl of enzyme solution, 10 μl of inhibitor solution(made up in water) and 50 μl of the appropriate 5 mM p-nitrophenylsubstrate (3.57 mM final conc.) made up in McIlvaine buffer at theoptimum pH for the enzyme.

The reactions were stopped with 0.4M glycine (pH 10.4) during theexponential phase of the reaction, which was determined at the beginningof the assay using blanks with water, which were incubated for a rangeof time periods to measure the reaction rate using 5 mM substratesolution. Endpoint absorbances were read at 405 nm with a Bioradmicrotitre plate reader (Benchmark). Water was substituted for theinhibitors in the blanks.

The enzymes tested are shown in Table 4.1, below. Enzyme Source pH Conc.Substrate α-D Saccharomyces cerevisiae 6.0 0.1 unit/mlPNP-α-D-glucopyranoside glucosidase (Baker's yeast), rice (Oryzasativa), Bacillus stearothermophilus β-D-glucosidase Almonds (Prunussp.) 5.0 0.2 unit/ml PNP-β-D-glucopyranoside α-D- Green coffee beans(Coffea sp.) 6.5   1 unit/ml PNP-α-D-galactopyranoside galactosidaseβ-D- Bovine liver 7.3 0.1 unit/ml PNP-β-D-galactopyranosidegalactosidase α-D- Jack beans (Canavalia ensiformis) 4.5 0.1 unit/mlPNP-α-D-mannopyranoside mannosidase α-L-fucosidase Bovine kidneyN-acetyl-β-D- Bovine kidney 4.2 0.1 unit/mlPNP-N-acetyl-β-D-glucosminide glucosaminidase 5 Naringinase Penecilliumdecumbens 4.0   1 unit/ml PNP-α-L-rhamnopyranoside

The compounds tested are shown in Table 4.2, below. Compound nameStructure Reference Castanospermine

20 Swainsonine

4 Casuarine

8 3,6,7-triepi-casuarine

12 3,6,7,7a-tetraepi- casuarine

21 3,7,7a-triepi-casuarine

22 3-epi-casuarine

14 3,7-diepi-casuarine

10 7-epi-casuarine

11

The results (% inhibition) for a number of different compounds (all at 1mg/ml) are shown in Table 4.3, below: Compound/ Enzyme 20 4 8 12 21 2214 10 11 gluc (yeast) −8 nd 64 2 −1 29 0 −2 11 gluc (rice) 77 nd 76 0 460 13 7 73 gluc (Bacillus) 6 nd 86 9 −2 87 12 −7 5 glucosidase 88 nd 0 644 52 56 5 30 galactosidase −3 nd 4 2 −3 −2 4 −11 1 galactosidase 16 nd0 6 3 52 6 24 35 mannosidase 9 74 5 8 1 −1 −4 8 10 fucosidase 3 nd −1−11 nd nd −2 5 25 Naringinase 39 nd −2 0 5 10 21 6 −4 N-acetyl-β-gluc 16nd 14 19 27 11 −1 −6 11

The results show that the profile of inhibition for the compounds of theinvention is quite different from that of castanospermine. None inhibitsmannosidase significantly (see also further data below). Some of thecompounds tested (e.g. 3,7-diepi-casuarine) do not significantly inhibitany of the enzymes tested.

Further studies showed that the K_(i) for casuarine (8) with yeastα-D-glucosidase was 217 μM (castanospermine not being inhibitory at aconcentration of 800 μM). The K_(i) for castanospermine (20) with almondβ-D-glucosidase was 9 μM (casuarine not being inhibitory at 800 μM).Moreover, casuarine also inhibited rabbit gut mucosa α-D-glucosidasewith an IC₅₀ value of 210 μM, as compared with an IC₅₀ value of 8 μM forcastanospermine. Both casuarine and castanospermine inhibited rabbitsmall intestine sucrose at a concentration of 700 μM. Castanosperminealso inhibited rabbit small intestine lactase and trehalase by over 50%at this concentration.

Example 5 Differential Inhibition of Mannosidase and Glucosidase

The glycosidase inhibitory profiles of swainsonine (45, casuarine (8)and casuarine glucoside (9) with respect to a mannosidase and aglucosidase were compared. The results (all at <0.1 mg/ml) are shown inTable 5.1, below. Glucosidase I Compound Mannosidase inhibitioninhibition Swainsonine (4) + − Casuarine (8) − + Casuarine glucoside (9)− +

Example 6 Treatment of Murine HSV-1 Infection

Mice were 3-4 weeks old female BALB/c. Mice were inoculated with 10⁴p.f.u. HSV-1 (SC16) using the neck skin method. This dose is sublethalbut produces clinical symptoms, including inflammation (measured byincrease in ear pinna thickness).

Mice were administered (100 ml i.p.) with one of two doses of casuarine(8) on day one and daily thereafter for 5 days. Group 1 received 15mg/kg in PBS, group 2 received 150 mg/kg in PBS. A negative controlgroup 3 were infected but received no casuarine. A positive controlgroup 4 were administered with famciclovir (via drinking water spiked at1 mg/ml for the same time period).

Mice were checked daily and samples were obtained from mice killed onselected days. The results are presented in Tables 6.1-6.3, below. TABLE6.1 Weight (% change) Group Day 1 2 3 4 −2 0 0 0 0 −1 0 3.1 3.2 1.3 9 15.6 5.8 4.6 13 2 5.6 5.2 6.5 14.5 3 8.6 7.1 9.3 18.8 4 7.4 5.8 9.8 18.15 8.6 8.4 10.5 21 6 9.2 9.7 12.4 23.9 7 7.4 7.7 11.1 21 8 9.3 8.4 13.723.9

TABLE 6.2 Group mean weight (g) Group Day 1 2 3 4 −2 16.2 15.5 15.3 13.8−1 0 16.7 16 15.5 15.1 1 17.1 16.4 16 15.6 2 17.1 16.3 16.3 15.8 3 17.616.6 16.7 16.4 4 17.4 16.4 16.8 16.3 5 17.6 16.8 16.9 16.7 6 17.7 1717.2 17.1 7 17.4 16.7 17 16.7 8 17.7 16.8 17.4 17.1 9 17.3 17.1 10 17.417.2 11 17.3 17.1 12 17.3 17.2

TABLE 6.3 Ear pinna thickness (mm²) Group Day 1 2 3 4 −2 0 0 0 0 −1 00.7 0.7 2.2 0 1 0 3.6 4.4 0 2 13.9 23.4 14.7 0 3 9 5.7 17.7 7 4 9 9.226.5 7 5 7.6 2.1 12.5 0 6 12.5 14.9 13.2 4 7 6.2 0 11 0 8 0 12.1 6.6 2.99 11.8 2.9 10 14 10.7 11 11 2.9 12 7.4 12.9 13 16.2 12.9

The results show the expected pattern of ear pinna thickness increase,peaking at day 4. Famvir almost completely negated the ear thicknessresponse. Casuarine at both doses tested also produced a reduction inear thickness.

Example 7 Control of Lung Metastasis in Mice

Mice (C57/bl6 under i/p ketamine anaesthesia) were challenged i/v (tailvein) with 5×10⁴ B16-F10 tumour cells in a final volume of 100 μl permouse on day 0. Test compounds (50 mg/kg in 200 μl sterile non-pyrogenicsaline) were administered s/c (right flank) on days 2 and 4. On day 14the mice were sacrifices and the lungs dissected and stained in Indianink solution (150 ml bidistilled water, 30 ml India Ink, 4 drops NH₄OH)for 10 minutes then fixed for at least 24 hr in Fakete's solution (90 ml37% formaldehyde, 900 ml 70% EtOH and 45 ml glacial acetic acid). Themetastases in the stained and fixed lungs could then be visualized,counted and photographed.

The results are shown below in Table 7.1, below. Compound Metastaticmorphology PBS (control) Metastasis over entire lung surface casuarine(8) Metastasis restricted to apical tip of lung 3-epi-casuarine (14)Metastasis restricted to apical tip of lung

Example 8 Effect on Glycosylation of Breast Cancer Cells

Cell Culture

MCF-7 cells (European Collection of Cell Cultures Ref. 86012803) weretaken from liquid nitrogen stock, thawed at room temperature andtransferred to 10 ml Dulbeccos Modified Eagle's Medium with Hams F12, 15mM Hepes and L-glutamine (DMEM: Cambrex. Cat. No. BE12-719F)supplemented with 10% v/v foetal calf serum (FCS: BioWest Labs Cat. No.S02755, Lot. No. S1800). The FCS was pre-filtered through a 0.2 μmsterile filter.

The cells were then centrifuged at 1,500 rpm in a Centaur bench-topcentrifuge and the supernatant removed. The cells were reconstituted infresh media and seeded into two T75 cm³ Nunclon tissue culture flasksand allowed to settle overnight at 37° C. in a 5% CO₂ incubator. Theflasks were wrapped in cling film to prevent cross-contamination and thefollowing day the media was changed to include the antibioticspenicillin and streptomycin as a precautionary measure against infection(at concentrations of 1 mg/cm³ and 5 mg/cm³, respectively).

The cells were allowed to grow near confluence and then split at a 1 in4 resuspension. The cells used for the experiments were of passagenumber 31. Two flasks of cells were prepared in media containing 20% v/vFCS with 10% dimethylsulphoxide and banked down into liquid nitrogen forlater use if necessary.

A total of 16 T25 cm³ flasks were used. Each flask was seeded with8.5×10⁵ cells/cm³ and 4 cm³ media added. The cells were allowed toadhere to the culture flask overnight. The following morning the flaskswere observed under the light microscope and the cells appeared 50-60%confluent. The cells from two of the flasks were harvested (see below)for the t=0 time point.

The remaining 14 flasks were available for testing with casuarine (8).Seven of these (untreated group) had their media changed to 7 cm³ offresh media containing 10% FCS, penicillin and streptomycin (as before),whilst the remaining seven were incubated with fresh media supplementedwith 0.75 mM casuarine (treated group).

Cells were harvested at t=1.5 hours, t=28 hours, t=62 hours and t=86hours.

Harvesting of Cells and Cell Counting

The cells were harvested using a non-enzymatic method. At each of thetime points the cells were viewed under the inverted light microscopeand the morphology evaluated. Before harvesting, the cells were washedwith sterile PBS, three times, 7 cm³ per wash. The cells were thenscraped from the flasks using a sterile cell scraper and transferred toGrenier tubes. The cells were quickly passed through a 21G2 gauge needleto disaggregate the cells. Cells were then pelleted by centrifugation at1500 g/5 min and resuspended in a known volume of PBS. The number ofcells was then counted in a haemocytometer and cell viability evaluatedby mixing 0.1 cm³ of each cell suspension with a drop of trypan bluesolution. Each of the cell pellets was frozen at −80° C. until glycanrelease and analysis.

Homogenisation

The cell pellets were placed in an iced water bath and allowed to thaw.The pellets were then homogenized in a total of 4 cm³ (made up to volumewith deionized water). An Ultraturrax T25 homogeniser was used for thispurpose, with the blade speed set to 22,500 rpm. The samples weremaintained on ice and 3 bursts, each of 10 sec, were applied with aperiod of approximately 1 min between each homogenisation step to allowthe froth the settle. The blade was washed carefully between each of thesamples to prevent sample cross-contamination. The homogenates werestored in 1 cm³ aliquots at −80° C. prior to the protein assay andglycan release.

Protein Assay

Evaluated using the BioRad protein assay according to the manufacturer'sinstructions. BSA was used as standard. Each of the homogenate sampleswas tested in duplicate using 100 μl aliquots from each time point

Glycan Release

For the time points of 62 hours and 86 hours the equivalent of 25 μg ofprotein was taken and dried for 3 hours on a centrifugal evaporator(without heating). For the earlier time points, whose proteinconcentration could not be assessed with the protein assay, 200 μl wastaken and dried down ready for glycan release. Release was confirmedusing 25 μg of fetuin from foetal calf serum.

Glycans were incubated at 37° C. overnight with N-glycosidase F (RocheBiosciences Cat. No. 1365185, Lot. No. 9280212/31) at a finalconcentration of 5U enzyme in 25 μl of sample all in 20 mM sodiumphosphate buffer pH 7.2. After the incubation step, the samples wereloaded onto prewashed and primed Ludger Clean E cartridges (Cat. No.LC-E10-A6). The glycans were eluted according to the manufacturer'sinstructions and dried by centrifugal evaporation overnight.

Glycan Labeling

The glycans were labeled by reductive amination, for 2 hours at 65° C.,according to the method described by Bigge et al. (1995) Anal. Biochem.230(2): 229-238. The incubation mixture was then “cleaned up” to removeany unconjugated fluorophore by spotting the samples onto Whatman 3MMpaper and running in a descending chromatography tank with a mobilephase of 4:1:1 butanol:ethanol:water overnight. Glycans were then elutedwith 0.5 cm³ methanol and 2×1 cm³ HPLC grade water then filtered througha 0.2 μm syringe top filter.

Analysis Using Normal Phase HPLC

The glycans were separated on a normal phase (hydrophilic interaction)HPLC column (LudgerSep N1 amide) 4.6×25 cm in size.

The basis of the separation is described in Guile et al. (1996) Anal.Biochem. 240(2): 210-226. The column was fitted to a Dionex BioLC systemwith autosampler and switching pump heads and in-line mixer. The columnwas maintained at 30° C. and the glycans detected using a Perkin ElmerLS30 fluorimeter with excitation λ=330 nm and emission λ=420 nm, thegain was set to 2. The buffer system used was the high salt system, withacetonitrile as buffer A and 0.25M ammonium formate pH4.4 as buffer B.Flow rate was maintained at 0.3 cm³/min throughout.

The protocol used is summarized below in Table 8.1, below. Time (min) %A % B Comment 0 80 20 Elution of N-linked glycans 132 47 53 135 0 100Elution of large charged glycans 142 0 100 145 80 20 Re-equilibration180 80 20 End of run

An 80 μl aliquot of each of the glycan mixtures was loaded onto thecolumn and the elution position compared, with reference to ahydrolysate of dextran.

Summary of Results and Conclusions

At the initial harvest point and the 28 hour time point, there was noobvious difference between the glycans released from the treated anduntreated cells (data not shown). However, at the 62 and 86 hour timepoints, the untreated cells showed a marked preponderance of largerN-linked glycans than their treated counterparts (data not shown). Inaddition, the overall signal (amount of fluorescently labeled glycan)was greater in the untreated group.

The results show that casuarine can inhibit glycan synthesis and/orN-linked glycosylation in breast cancer cells.

Example 9 Effect on Glucose Transport

The effect of casuarine (8) and castanospermine (20) on the initial rateof Na⁺-dependent D-glucose uptake into ovine intestinal brush bordermembrane vesicles was examined in a competition assay with labeledD-glucose. The results are shown in Table 9.1, below: Compound ReferenceGlucose uptake (pmol s⁻¹mg⁻¹) None (control) 240 Casuarine 8 265Castanospermine 20 225

It can be seen that glucose transport was slightly inhibited bycastanospermine but slightly stimulated by casuarine.

Example 10 Increasing the Th1:Th2 Response Ratio in a Non-HealingLeishmaniasis Model

Leishmaniasis is a classic model of a Th1 disease: non-healing cutaneouslesions arise from an undesirable polarization of the immune responsewhich becomes heavily Th2-skewed.

In order to study the ability of the compounds of the invention toincrease the Th1:Th2 response ratio in this disease model (and sopromote a healing Th1 response), spleen cells from Leishmania majorinfected BALB/c mice having a non-healing cutaneous infection werestimulated with parasite antigen (Table 10.1) or polyclonally withanti-CD3 (Table 10.2) in the presence of 3,7-diepi-casuarine (10). TABLE10.1 Reversal of the inability of T-cells to produce IFN-y in anon-healing mouse model Treatment IFN-y (ng/ml) None (control) ˜0.5 L.major Ag ˜0.5 3,7-diepi-casuarine (10) ˜0.5 3,7-diepi-casuarine (10) +L. major Ag  5.5

TABLE 10.2 Downregulation of Th2 cytokine response in a non-healingmouse model Treatment IL-5 (pg/ml) None (control) 50 aCD3 2403,7-diepi-casuarine (10) + aCD3 150

It can be seen that the presence of 3,7-diepi-casuarine (10) enhancesIFN-γ (associated with a healing Th1 response) whilst suppressing theTh2 response (via downregulation of the Th2 cytokine IL-5). TheTh2-skewed immune response profile associated with a non-healing diseasewas clearly reversed ex vivo by 3,7-diepi-casuarine (10).

Example 11 Synthesis of 3,7-diepi-casuarine (10)

General Experimental

All reactions were carried out under an atmosphere of argon at roomtemperature using anhydrous solvents unless otherwise stated. Anhydroussolvents were purchased from Fluka Chemicals and were used as supplied.Reagents were supplied from Aldrich, Fluka and Fisher and were used assupplied. Thin layer chromatography (Tlc) was performed on aluminiumsheets pre-coated with Merck 60 F₂₅₄ silica gel and were visualisedunder ultra-violet light and staining using 6% phosphomolybdic acid inethanol. Silica gel chromatography was carried out using Sorbsil C6040/60 silica gel under a positive atmosphere. Amberlite IR-120, stronglyacidic ion-exchange resin was prepared by soaking the resin in 2Mhydrochloric acid for at least two hours followed by elution withdistilled water until the eluant reached pH 5. Dowex: 50WX8-100 wasprepared by soaking the resin with 2M hydrochloric acid for at least twohours followed by elution with distilled water until neutral. Infraredspectra were recorded on a Perkin-Elmer 1750 IR Fourier Transformspectrophotometer using thin films on sodium chloride plates. Onlycharacteristic peaks are recorded. Optical rotations were measured on aPerkin-Elmer 241 polarimeter with a path length of 1 dm. Concentrationsare quoted in g/100 mL. Nuclear magnetic resonance spectra were recordedon a Bruker DQX 400 spectrometer in the stated deuterated solvent. Allspectra were recorded at ambient temperature. Chemical shifts (δ) arequoted in ppm and are relative to residual solvent as standard. Protonspectra (δ_(H)) were recorded at 400 MHz and carbon spectra (δ_(C)) at100 MHz.

2,3:5,6:7,8-Tri-O-isopropylidene-D-erythro-L-talo-octono-1,4-lactone(Qc)

5,6:7,8-Di-O-isopropylidene-D-erythro-L-galacto-octono-1,4-lactone (Qb)

Sodium cyanide (7.02 g, 142 mmol) was added to a stirred solution ofD-glycero-D-gulo-heptose (Qa, 21 g, 100 mmol) in water (300 ml). Thereaction mixture was stirred at room temperature for 48 h, heated atreflux for 48 h and passed through a column containing Amberlite IR-120(strongly acidic ion-exchange resin, 300 ml). The eluent wasconcentrated under reduced pressure and the residue dried in vacuo for24 hours. The resulting foam was treated with acetone (500 ml) andsulphuric acid (5.4 ml) in the presence of anhydrous copper sulphate (10g, 62 mmol) at room temperature for 48 h. T.l.c analysis indicated thepresence of two major products (ethyl acetate:cyclohexane, 1:1; R_(f)0.72, 0.18). The reaction mixture was filtered and the filtrate wastreated with sodium bicarbonate (50 g) for 24 h at room temperature.Solid residues were removed by filtration and the filtrate wasconcentrated under reduced pressure. The resulting crude yellow syrupwas purified by silica gel chromatography providing2,3:5,6:7,8-tri-O-isopropylidene-D-erythro-L-talo-octono-1,4-lactone Qcas a colourless syrup (R_(f) 0.72; 7.672 g; 21%;) and5,6:7,8-di-O-isopropylidene-D-erythro-L-galacto-octono-1,4-lactone Qb asa clear oil (R_(f) 0.18; 8.105 g; 25%)2,3:5,6:7,8-tri-O-isopropylidene-D-erythro-L-talo-octono-1,4-lactone Qc: δ_(H) (CDCl₃) 1.29, 1.33, 1.35, 1.38, 1.42, 1.48 (6×s, 18H,3×C(CH₃)₂), 3.93-3.99 (m, 2H, H-8_(a), H-7), 4.03-4.07 (m, 2H, H-5,H-6), 4.15 (dd, 1H, J_(8a,8b) 8.7 J_(8b,7) 6.1, H-8_(b)), 4.75-4.78 (m,3H, H-2, H-3, H-4); δ_(C) (CDCl₃) 25.23, 25.51, 26.00, 26.71, 26.73,27.16 (3×C(CH₃)₂), 67.93, 74.93, 76.33, 76.69, 78.65, 79.40, 80.06,109.95, 110.72, 113.19, 174.27; ν_(max) (film) 1793.5,6:7,8-di-O-isopropylidene-D-erythro-L-galacto-octono-1,4-lactone Qb :δ_(H) (d₆-acetone) 1.28, 1.32, 1.34, 1.35 (4s, 12H, 2×C(CH₃)₂), 3.92(1H, m, H-8_(a)), 3.98 (m, 1H, H-7), 4.14 (m, 2H, H-5, H-8_(b)),4.23-4.25 (m, 2H, H-4, H-6), 4.35-4.40 (m, 2H, H-2, H-3); δ_(C)(d₆-acetone) 25.31, 25.87, 26.72, 27.31, 68.06, 75.15, 75.23, 77.51,78.05, 78.41, 79.01, 110.06, 110.31, 174.25; ν_(max) (film) 1793, 3541.

2,3:5,6-Di-O-isopropylidene-D-erythro-L-talo-octono-1,4-lactone Qd

A solution of2,3:5,6:7,8-tri-O-isopropylidene-D-erythro-L-talo-octono-1,4-lactone(Qc, 3.8 g, 10.6 mmol) was treated with acetic acid:water (2:3, 100 ml)at 50° C. for 2 h. T.l.c analysis (ethyl acetate:cyclohexane, 1:1)indicated the disappearance of the starting material (R_(f) 0.72) andthe presence of a more polar compound (R_(f) 0.15). The solvent wasremoved under reduced pressure and the residue was purified by silicagel chromatography (ethyl acetate:cyclohexane, 1:1 to 3:1) yielding2,3:5,6-di-O-isopropylidene-D-erythro-L-talo-octono-1,4-lactone Qd as aclear oil (3.23 g, 94%): δ_(H) (CD₃OD) 1.28, 1.38, 1.43 (3×s, 12H,2×C(CH₃)₂), 3.59 (dd, 1H, J_(8a,7) 5.40 J_(8a,8b) 11.41, H-8_(a)),3.66-3.69 (m, 1H, H-7), 3.74 (dd, 1H, J_(8ab,7) 2.90 Hz, H-8_(b)), 4.01(app t, 1H, J_(6,7) 7.62 Hz, H-6); 4.24 (dd, 1H, J_(5,6) 8.17 Hz J_(5,4)0.89 Hz, H-5), 4.79-4.81 (m, 2H, H-3, H-4), 4.89-4.91 (m, 1H, H-2);δ_(C) (CD₃OD) 24.62, 25.42, 26.05, 26.49, 63.86, 73.81, 75.40, 75.91,79.18, 70.90, 80.78, 110.53, 113.09, 175.76; ν_(max) (film) 1791, 3478;[α]_(D) −35.7 (c 1, CHCl₃).

8-O-tert-Butyldimethylsilyl-2,3:5,6-di-O-isopropylidene-D-erythro-L-talo-octono-1,4-lactoneQe

To a solution of2,3:5,6-di-O-isopropylidene-D-erythro-L-talo-octono-1,4-lactone (Qd,3.18 g, 10 mmol) in N,N-dimethylformamide (40 ml) was addedtert-butyldimethylsilyl chloride (1.808 g, 12 mmol) and imidazole (1.361g, 20 mmol). The reaction mixture was stirred at room temperature for 16h after which t.l.c. analysis (ethyl acetate:cyclohexane, 1:1) showed nostarting material (R_(f) 0.15) and the formation of one major product(R_(f 0.63)). The solvent was removed under reduced pressure and theresidue was partitioned between ethyl acetate and brine. The aqueouslayer was extracted with ethyl acetate and the combined organic layerswere dried (MgSO₄), filtered and the solvent removed. The resulting paleoil was purified by silica gel chromatography (ethylacetate:cyclohexane, 0:1 to 1:2) to give8-O-tert-butyldimethylsilyl-2,3:5,6-di-O-isopropylidene-D-erythro-L-talo-octono-1,4-lactoneQe as a clear oil (3.612 g, 85%): δ_(H) ((CDCl₃) 0.04 (br s, 6H, 2×CH₃),0.86 (s, 9H, C(CH₃)₃), 1.23, 1.30, 1.32, 1.41 (4×s, 12H, 2×C(CH₃)₂),3.63-3.67 (m, 2H, H-8_(a), H-7), 3.76 (br d, 1H, H-8_(b)), 3.96 (app t,J_(6,7) 8.21 J_(6,5) 7.98, H-6), 4.08 (br d, 1H, H-5), 4.72 (br s, 2H,H-2, H-3), 4.78 (br s, 1H, H-4); δ_(C) (CDCl₃) −5.52, −5.45, 18.25,25.51, 25.80, 25.93, 26.68, 27.18, 63.95, 72.97, 74.88, 74.93, 78.71,79.63, 79.87, 110.34, 113.00, 174.42; ν_(max) (film) 1794, 3570; [α]_(D)−20.1 (c 1, CHCl₃).

7-Azido-8-O-tert-butyldimethylsilyl-7-deoxy-2,3:5,6-di-O-isopropylidene-L-threo-L-talo-octono-1,4-lactoneQf

A solution of8-O-tert-butyldimethylsilyl-2,3:5,6-di-o-isopropylidene-D-erythro-L-talo-octono-1,4-lactone(Qe, 3.5 g, 8.2 mmol) in a pyridine:dichloromethane mixture (1:4, 25 ml)was cooled to −30° C. Trifluoromethanesulfonic anhydride (3.5 g, 2.09ml, 12.4 mmol) was added portion-wise and the mixture was stirred for 2h. T.l.c analysis (ethyl acetate:cyclohexane, 1:3) indicated thedisappearance of starting material (R_(f) 0.38) and the presence of aless polar product (R_(f) 0.48). The reaction mixture was concentratedunder reduced pressure and the residue was partitioned between ethylacetate and 0.5 M hydrochloric acid. The organic layer was washed withbrine, dried (MgSO₄), filtered and concentrated under reduced pressure.The resulting crude pale orange residue was treated with sodium azide(807 mg, 12.4 mmol) in N,N-dimethylformamide (25 ml) for 16 h. T.l.c.analysis (ethyl acetate:cyclohexane, 1:4) indicated the disappearance ofthe intermediate triflate (R_(f) 0.42) and the presence of a more polarcompound (R_(f) 0.40). The reaction solvent was removed in vacuo and theresidue was partitioned between ethyl acetate and brine. The aqueouslayer was extracted with ethyl acetate and the combined organic layerswere dried (MgSO₄), filtered and concentrated in vacuo. The resultingcrude residue was purified by silica gel chromatography (ethylacetate:cyclohexane, 0:1 to 1:4) providing7-azido-8-O-tert-butyldimethylsilyl-7-deoxy-2,3:5,6-di-O-isopropylidene-L-threo-L-talo-octono-1,4-lactoneQf as a colourless oil (3.026 g, 81%): δ_(H) (CDCl₃) 0.11 (2×s, 6H,2×CH₃), 0.91 (s, 9H, C(CH₃)₃), 1.30, 1.38, 1.41, 1.47 (4×s, 12H,2×C(CH₃)₂), 3.41-3.45 (m, 1H, H-7), 3.87 (dd, 1H, J_(8a,7) 5.37 HzJ_(8a,8b) 10.81 Hz, H-8_(a)), 3.92 (dd, 1H, J_(8b,7) 7.32 Hz, H-8_(b)),4.19-4.24 (m, 2H, H-5, H-6), 4.61 (br s, 1H, H-4), 4.75-4.79 (m, 2H,H-2, H-3); δ_(C) (CDCl₃) −5.59, −5.56, 18.14, 25.54, 25.73, 26.09,26.71, 26.98, 61.61, 63.19, 67.94, 74.84, 74.94, 75.47, 78.36, 78.66,110.90, 113.37, 174.02; ν_(max) (film) 1796, 2111; [α]_(D)+36.7 (c 1,CHCl₃).

7-Azido-8-O-tert-butyldimethylsilyl-7-deoxy-2,3:5,6-di-O-isopropylidene-L-threo-L-talo-octitolQg

7-azido-8-O-tert-butyldimethylsilyl-7-deoxy-2,3:5,6-di-O-isopropylidene-L-threo-L-talo-octono-1,4-lactone(Qf, 3.00 g, 6.6 mmol) was dissolved in tetrahydrofuran (40 ml) and wascooled to 0° C. Lithium borohydride (216 mg, 9.9 mmol) was added and themixture was stirred at 0° C. to room temperature for 24 h. T.l.c.analysis (ethyl acetate:cyclohexane, 1:1) indicated the disappearance ofthe starting material (R_(f) 0.76) and the presence of a more polarcompound (R_(f) 0.45). The reaction was quenched through the addition ofammonium chloride (sat. aq.) and the partitioned between ethyl acetateand brine. The aqueous layer was extracted with ethyl acetate (2×) andthe combined organic layers were dried (MgSO₄), filtered and the solventremoved. The resulting crude residue was purified by silica gelchromatography (ethyl acetate:cyclohexane, 1:3 to 1:1) affording7-azido-8-O-tert-butyldimethylsilyl-7-deoxy-2,3:5,6-di-O-isopropylidene-L-threo-L-talo-octitolQg as a colourless syrup (2.476 g, 82%): δ_(H) (CDCl₃) 0.10 (s, 6H,2×CH₃), 0.91 (s, 9H, C(CH₃)₃), 1.36, 1.41, 1.42, 1.48 (4×s, 12H,2×C(CH₃)₂), 3.43-3.47 (m, 1H, H-7), 3.66 (br d, 1H, H-4), 3.79-3.92 (m,4H, H-1, H-1_(a), H-8, H-8_(a)), 4.10-4.14 (m, 2H, H-2, H-3), 4.30-4.38(m, 2H, H-5, H-6); δ_(C) (CDCl₃) −5.61, −5.51, 18.14, 25.18, 25.71,26.87, 27.07, 27.86. 60.65, 62.39, 63.66. 67.62, 75.90. 76.91, 77.18,77.49, 108.63, 110.16; ν_(max) (film) 2109, 3536; [α]_(D)+46.6 (c 1,CHCl₃).

7-Azido-8-O-tert-butyldimethylsilyl-7-deoxy-2,3:5,6-di-O-isopropylidene-1,4-di-O-methanesulphonyl-L-threo-L-talo-octitolQh

7-Azido-8-O-tert-butyldimethylsilyl-7-deoxy-2,3:5,6-di-O-isopropylidene-L-threo-L-talo-octitol(Qg, 2.4 g, 5.3 mmol) was dissolved in pyridine (20 ml) and was added toa solution of 4-dimethylamino pyridine (64 mg, 0.53 mmol) andmethanesulfonyl chloride (4.814 g, 3.253 ml, 42 mmol) in pyridine (20ml) and stirred for 2 h. T.l.c analysis (ethyl acetate:cyclohexane, 1:2,double elution) revealed the disappearance of starting material (R_(f)0.33) and the presence of a more hydrophobic product (R_(f) 0.43). Thesolvent was removed under educed pressure and the residue waspartitioned between ethyl acetate and brine. The aqueous layer wasextracted with ethyl acetate and the combined organic layers were dried(MgSO₄), filtered and concentrated under reduced pressure. The resultingcrude residue was purified by silica gel chromatography (ethylacetate:cyclohexane, 1:2) giving7-azido-8-O-tert-butyldimethylsilyl-7-deoxy-2,3:5,6-di-O-isopropylidene-1,4-di-O-methanesulfonyl-L-threo-L-talo-octitolQh as a colourless oil (2.973 g, 92%): δ_(H) (CDCl₃) 0.11, 0.12 (2×s,6H, 2×CH₃), 0.91 (s, 9H, C(CH₃)₃), 1.41, 1.44, 1.46, 1.56 (4×s, 12H,2×C(CH₃)₂), 3.08 (s, 3H, SO₂CH₃), 3.21 (s, 3H, SO₂CH₃), 3.49 (ddd, 1H,J_(7,6) 2.82 Hz, J_(7,8) 5.46 Hz, J_(7,8a) 7.94 Hz, H-7), 3.87-3.97 (m,2H, H-8, H-8_(a)), 4.19 (dd, 1H, J_(6,5) 2.30 Hz, H-6), 4.24-4.31 (m,2H, H-1, H-5), 4.36 (dd, 1H, J_(3,4) 2.96 Hz, J_(3,2) 6.62 Hz, H-3),4.49-4.53 (m, 1H, H-2), 4.69 (dd, 1H. J_(1a,2) 2.39 Hz, J_(1a,1) 10.83Hz, H-1_(a)), 5.11 (app t, 1H, H-4); δ_(C) (CDCl₃) −5.56, 18.18, 25.76,26.24, 26.78, 26.89, 27.56, 37.75, 39.02, 60.90, 63.57, 70.44, 76.00,76.07, 76.46, 77.18, 77.32, 109.01, 110.68; ν_(max) (film) 2113; [α]_(D)−16.2 (c 1, CHCl₃).

7-Azido-7-deoxy-1,4-di-O-methanesulphonyl-L-threo-L-talo-octitol Qi

7-Azido-8-O-tert-butyldimethylsilyl-7-deoxy-2,3:5,6-di-O-isopropylidene-1,4-di-O-methanesulfonyl-L-threo-L-talo-octitol(Qh, 2.90 g, 4.7 mmol) was treated with a trifluroacetic acid:watermixture (1:1, 40 ml) for 3 h. T.l.c. analysis (ethyl acetate) showed thedisappearance of starting material (R_(f) 0.9) and the presence of amore polar product (R_(f) 0.12). The solvent was removed under reducedpressure and the residue was co-evaporated with toluene and dried undervacuum. Purification by silica gel chromatography (ethylacetate:cyclohexane, 1:1 to 1:0) yielded7-azido-7-deoxy-1,4-di-O-methanesulphonyl-L-threo-L-talo-octitol Qi as acolourless oil (1.677 g, 85%): δ_(H) (CD₃OD) 3.12 (s, 3H, SO₂CH₃), 3.21(s, 3H, SO₂CH₃), 3.61-3.71 (m, 2H, H-7, H-8), 3.78-3.82 (m, 2H, H-6,H-8_(a)), 3.98-4.05 (m, 2H, H-2, H-3), 4.11-4.13 (m, 1H, H-5), 4.34 (dd,1H, J_(1,2) 4.87 Hz, J_(1,1a) 10.44 Hz, H-1), 4.45 (dd, 1H, J_(1,2) 1.87Hz, H-1_(a)), 5.00 (dd, 1H, J_(4,3) 1.91 Hz, J_(4,5) 6.15 Hz, H-4);δ_(C) (CD₃OD) 36.17, 38.11, 61.84, 66.62, 69.09, 70.33, 70.45, 71.08,72.55, 86.41; ν_(max) (film) 2113; [α]_(D) −9.1 (c 1, H₂O).

(1R,2R,3S,6S,7R,7aR)-3-(Hydroxymethyl)-1,2,6,7-tetrahydroxypyrrolizidineQi

[3,7-diepi-Casuarine]

7-Azido-7-deoxy-1,4-di-O-methanesulphonyl-L-threo-L-talo-octitol (Qi,1.6 g, 3.78 mmol) was dissolved in water (30 ml) and was treated with10% palladium on carbon (400 mg) under an atmosphere of hydrogen for 16h. T.l.c analysis (ethyl acetate:methanol, 9:1) indicated thedisappearance of starting material (R_(f) 0.75) and the presence of amore polar product (R_(f) 0.05). Palladium was removed by filtration andthe filtrate was treated with sodium acetate (930 mg, 11.34 mmol) at 60°C. for 16 h. The reaction mixture was cooled and the solvent removed invacuo. The crude brown oil was purified by ion-exchange chromatography(Dowex 50WX8-100, eluting with 2M ammonium hydroxide) to afford(1R,2R,3S,6S,7R,7aR)-3-(hydroxymethyl)-1,2,6,7-tetrahydroxypyrrolizidine[3,7-diepi-Casuarine] Qj as a brown glass (671 mg, 87%): δ_(H) (D₂O)2.81-2.92 (m, 2H, H-5, H-5_(a)), 3.16 (dd, 1H, J_(3,2) 5.91 Hz, J_(3,8)10.74 Hz, H-3), 3.30 (app t, 1H, J 3.78 Hz, H-7_(a)), 3.76 (dd, 1H,J_(8,8a) 6.35 Hz, H-8), 3.87 (dd, 1H, H-8_(a)), 4.01 (d, 1H, J_(2,1)3.55 Hz, H-2), 4.04-4.12 (m, 2H, H-6, H-7), 4.29 (app t, 1H, H-1); δ_(C)(D₂O) 49.32, 57.29, 63.78, 70.41, 72.59, 72.65, 74.47, 78.25; [α]_(D)−21.1 (c 0.5, H₂O).

Equivalents

The foregoing description details presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare intended to be encompassed within the claims appended hereto.

1-44. (canceled)
 45. A method for treating a disease associated with adeleterious immune response comprising administering to a patient inneed of such treatment a therapeutically effective amount of apolyhydroxylated pyrrolizidine compound of formula:

wherein R is selected from the group comprising hydrogen, straight orbranched, unsubstituted or substituted, saturated or unsaturated acyl,alkyl, alkenyl, alkynyl and aryl groups, or a pharmaceuticallyacceptable salt or acyl derivative thereof.
 46. A method according toclaim 45 wherein the pyrrolizidine compound has the formula:

wherein R is selected from the group comprising hydrogen, straight orbranched, unsubstituted or substituted, saturated or unsaturated acyl,alkyl, alkenyl, alkynyl and aryl groups, or a pharmaceuticallyacceptable salt or acyl derivative thereof.
 47. A method according toclaim 45 wherein the pyrrolizidine compound is a glycosidase inhibitor.48. A method according to claim 45 wherein the pyrrolizidine compound,when administered in vivo, modifies one or more of: (a) tumour cellglycosylation; (b) viral protein glycosylation; (c) cell-surface proteinglycosylation; (d) bacterial cell walls and (e) cytokine releaseactivity, by stimulation of secretion of one or more cytokine.
 49. Amethod according to claim 45 wherein the pyrrolizidine compound is anacyl derivative.
 50. A method according to claim 49 wherein thepyrrolizidine acyl derivative is chosen from a peracylated derivative, aderivative that is acylated at C-3 hydroxymethyl; a derivative that isacylated at C-6; and a derivative that is acylated at C-3 hydroxymethyland C-6.
 51. A method according to claim 49 wherein the acyl derivativeis an alkanoyl derivative selected from acetyl, propanoyl and butanoyl.52. A method according to claim 45 wherein R is a saccharide moiety. 53.A method according to claim 52 wherein the saccharide moiety is aglucoside or arabinoside moiety.
 54. A method according to claim 45wherein the pyrrolizidine compound is chosen from: (a)1R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)-1,2,6,7-tetrahydroxypyrrolizidine(casuarine), wherein R is hydrogen and having the formula:

(b) a casuarine glycoside; (c) casuarine-6-α-D-glucoside of the formula:

(d) 6-O-butanoylcasuarine; (e) 3,7-diepi-casuarine; (f) 7-epi-casuarine;(g) 3,6,7-triepi-casuarine; (h) 6,7-diepi-casuarine; (i)3-epi-casuarine; (j) 3,7-diepi-casuarine-6-α-D-glucoside; (k)7-epi-casuarine-6-α-D-glucoside; (l)3,6,7-triepi-casuarine-6-α-D-glucoside;(m)6,7-diepi-casuarine-6-α-D-glucoside; (n)3-epi-casuarine-6-α-D-glucoside, and a pharmaceutically acceptable saltor derivative of any of (a)-(n).
 55. A method according to claim 45wherein said polyhydroxylated pyrrolizidine compound is administered incombination with an additional therapeutic agent chosen from one or moreof: (a) an immunostimulant; (b) a cytotoxic agent; (c) an antimicrobialagent; (d) an antiviral agent; and (e) a primed dendritic cell.
 56. Amethod according to claim 46 wherein said disease is chosen from (a) aproliferative disorder; (b) a Th1-related disease or disorder; (c) aTh2-related disease or disorder; (d) a bacterial infection; (e) a viralinfection; (f) a prion, fungal, protozoan or metazoan infection; and (g)a disease associated with an intracellular pathogen.
 57. A methodaccording to claim 56 wherein said viral infection is selected fromrespiratory syncytial virus (RSV), hepatitis B virus (HBV),Epstein-Barr, hepatitis C virus (HCV), herpes simplex type 1 and 2,herpes genitalis, herpes keratitis, herpes encephalitis, herpes zoster,human immunodeficiency virus (HIV), influenza A virus, hantann virus(hemorrhagic fever), human papilloma virus (HPV) and measles.
 58. Amethod for immunomodulation comprising administering to a patient inneed of such treatment a composition containing a therapeuticallyeffective amount of a a polyhydroxylated pyrrolizidine compound offormula:

wherein R is selected from the group comprising hydrogen, straight orbranched, unsubstituted or substituted, saturated or unsaturated acyl,alkyl, alkenyl, alkynyl and aryl groups, or a pharmaceuticallyacceptable salt or acyl derivative thereof.
 59. The method of claim 58wherein said composition comprises a herbal medicine.
 60. A methodaccording to claim 59 wherein said herbal medicine derives from one ormore plant species sources selected from: (a) a member of the taxonMyrtaceae (b) a member of the taxon Casuarinaceae; (c) a combination oftwo or more plant species selected from both of the taxons of (a) and(b).
 61. A method according to claim 58 wherein the Th1:Th2 responseratio is increased.
 62. A method for immunomodulation according to claim58 chosen from (a) haemorestoration; (b) haemoablative immunotherapy;(c) alleviation of immunosuppression; (d) cytokine stimulation; (e)vaccination, wherein the pyrrolizidine compound acts as an adjuvant; (f)vaccination with a dendritic cell vaccine wherein the dendritic cellsare contacted with the pyrrolizidine compound; (g) administration ofdendritic cells in the treatment or prophylaxis of autoimmune disorders,wherein the dendritic cells are contacted with the pyrrolizidinecompound; (h) wound healing; (i) stimulating the innate immune response;(j) boosting the activity of endogenous NK cells; (k) inducing,potentiating or activating one or more cytokines in vivo; and (l)providing chemoprotection to a patient undergoing chemotherapy.
 63. Avaccine comprising (1) a polyhydroxylated pyrrolizidine compound offormula:

wherein R is selected from the group comprising hydrogen, straight orbranched, unsubstituted or substituted, saturated or unsaturated acyl,alkyl, alkenyl, alkynyl and aryl groups; in combination with (2) anantigen, said pyrrolizidine compound being present in an amountsufficient to produce an adjuvant effect on vaccination.